PUBLICATIONS
The cutting-edge work done at Proteros are often published with our collaborators in academia and industry, highlighting the trust that our collaborators place in us to help them REACH RIGHT FASTER.
Discovery and Preclinical Profile of ALG-055009, a Potent and Selective Thyroid Hormone Receptor Beta (THR-β) Agonist for the Treatment of MASH
Author: Koen Vandyck, Jerome Deval, David C. McGowan, Xuan G. Luong, Sarah K. Stevens, Andreas Jekle, Kusum Gupta, Dinah Misner, Sushmita Chanda, Vladimir Serebryany, Michael Welch, Caroline Williams, Klaus Maskos, Alfred Lammens, Antitsa D. Stoycheva, Lawrence M. Blatt, Leonid N. Beigelman, Julian A. Symons, Pierre Raboisson
Publication with: Proteros Biostructures GmbH, ALIGOS therapeutics
Citation:
Abstract:
Agonists of thyroid hormone receptor β (THR-β) decreased LDL cholesterol (LDL-C) and triglyceride (TG) levels in human clinical trials for patients with dyslipidemia. The authors present the highly potent and selective compound ALG-055009 (14) as a potential best in class THR-β agonist. The high metabolic stability and good permeability translated well in vivo to afford a long in vivo half-life pharmacokinetic profile with limited liability for DDI, and it overcomes certain drawbacks seen in recent clinical candidates.
Cryo-EM Structures of CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 Complexes
Author: Dirk Dedden, Julius Nitsche, Elisabeth V. Schneider, Maren Thomsen, Daniel Schwarz, Birgitta Leuthner, Ulrich Grädler
Publication with: Proteros Biostructures GmbH, Merck KGaA
Citation: Dedden, D., Nitsche, J., Schneider, E. W., Thomsen, M., Schwarz, D., Leuthner, B., & Grädler, U. (2024). Cryo-EM structures of CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 complexes. Journal of Molecular Biology, 168483
Abstract:
RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) and X-ray structures of BRAF in different conformational states as inactive or active complexes with KRAS, 14-3-3 and MEK1. In this study, we have solved the first cryo-EM structures of CRAF2/14-3-32 at 3.4 Å resolution and CRAF2/14-3-32/MEK12 at 4.2 Å resolution using CRAF kinase domain expressed as constitutively active Y340D/Y341D mutant in insect cells. The overall architecture of our CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 cryo-EM structures is highly similar to corresponding BRAF structures in complex with 14-3-3 or 14-3-3/MEK1 and represent the activated dimeric RAF conformation. Our CRAF cryo-EM structures provide additional insights into structural understanding of the activated CRAF2/14-3-32/MEK12 complex.
Physicochemical and functional comparability between the proposed biosimilar rituximab GP2013 and originator rituximab
Author: Visser J, Feuerstein I, Stangler T, Schmiederer T, Fritsch C, Schiestl M
Publication with: Sandoz pharma and Hexal
Citation: Physicochemical and functional comparability between the proposed biosimilar rituximab GP2013 and originator rituximab. BioDrugs. 2013 Oct;27(5):495-507.
Abstract:
Background: Regulatory approval for a biosimilar product is provided on the basis of its comparability to an originator product. A thorough physicochemical and functional comparability exercise is a key element in demonstrating biosimilarity. Here we report the characterization of a proposed biosimilar rituximab (GP2013) and originator rituximab.
Structures, functions, and adaptations of the human LINE-1 ORF2 protein
Author: Baldwin, E.T., van Eeuwen, T., Hoyos, D
Publication with: ROME Therapeutics, Boston
Citation: Baldwin, E.T., van Eeuwen, T., Hoyos, D. et al. Structures, functions, and adaptations of the human LINE-1 ORF2 protein. Nature (2023).
Abstract:
The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one third of the human genome through a “copy-and-paste” mechanism catalyzed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5, and aging6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p ‘core’ (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-EM in multiple conformational states. Our analyses reveal two novel folded domains, extensive contacts to RNA templates, and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry reveal that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signaling via cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which likely explains cytosolic priming. Additional biochemical activities including processivity, DNA-directed polymerization, non-templated base addition, and template switching together allow us to propose an updated L1 insertion model. Finally, our evolutionary analysis reveals structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on L1 evolutionary history, and enable rational drug development targeting L1.
Human endogenous retrovirus-K (HERV-K) reverse transcriptase (RT) structure and biochemistry reveals remarkable similarities to HIV-1 RT and opportunities for HERV-K-specific inhibition
Author: Baldwin ET, Götte M, Tchesnokov EP, Arnold E, Hagel M, Nichols C, Dossang P, Lamers M, Wan P, Steinbacher S, Romero DL
Publication with: ROME Therapeutics, Boston
Citation: Proc Natl Acad Sci U S A . 2022 Jul 5;119(27):e2200260119. doi: 10.1073/pnas.2200260119. Epub 2022 Jun 30.
Abstract:
Human endogenous retroviruses (HERVs) comprise nearly 8% of the human genome and are derived from ancient integrations of retroviruses into the germline. The biology of HERVs is poorly defined, but there is accumulating evidence supporting pathological roles in diverse diseases, such as cancer, autoimmune, and neurodegenerative diseases. Functional proteins are produced by HERV-encoded genes, including reverse transcriptases (RTs), which could be a contributor to the pathology attributed to aberrant HERV-K expression. To facilitate the discovery and development of HERV-K RT potent and selective inhibitors, we expressed active HERV-K RT and determined the crystal structure of a ternary complex of this enzyme with a double-stranded DNA substrate. We demonstrate a range of RT inhibition with antiretroviral nucleotide analogs, while classic nonnucleoside analogs do not inhibit HERV-K RT. Detailed comparisons of HERV-K RT with other known RTs demonstrate similarities to diverse RT families and a striking similarity to the HIV-1 RT asymmetric heterodimer. Our analysis further reveals opportunities for selective HERV-K RT inhibition.
Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics
Author: Schubert J, Schulze A, Prodromou C, Neuweiler H.
Publication with: Julius-Maximilians-University Würzburg
Citation: Nat Commun. 2021 Nov 29;12(1):6964. doi: 10.1038/s41467-021-27286-5.
Abstract:
Many proteins are molecular machines, whose function is dependent on multiple
conformational changes that are initiated and tightly controlled through
biochemical stimuli. Their mechanistic understanding calls for spectroscopy that
can probe simultaneously such structural coordinates. Here we present two-colour
fluorescence microscopy in combination with photoinduced electron transfer (PET)
probes as a method that simultaneously detects two structural coordinates in
single protein molecules, one colour per coordinate. This contrasts with the
commonly applied resonance energy transfer (FRET) technique that requires two
colours per coordinate. We demonstrate the technique by directly and
simultaneously observing three critical structural changes within the Hsp90
molecular chaperone machinery. Our results reveal synchronicity of
conformational motions at remote sites during ATPase-driven closure of the Hsp90
molecular clamp, providing evidence for a cooperativity mechanism in the
chaperone's catalytic cycle. Single-molecule PET fluorescence microscopy opens
up avenues in the multi-dimensional exploration of protein dynamics and
allosteric mechanisms.
Molecular basis of human ATM kinase inhibition
Author: Stakyte K, Rotheneder M, Lammens K, Bartho JD, Grädler U, Fuchß T, Pehl U, Alt A, van de Logt E, Hopfner KP.
Publication with: Ludwig-Maximilians-Universität
Citation: Nat Struct Mol Biol. 2021 Oct;28(10):789-798. doi: 10.1038/s41594-021-00654-x. Epub 2021 Sep 23.
Abstract:
Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in
initiation of the DNA damage response following DNA double-strand breaks. ATM
inhibition is a promising approach in cancer therapy, but, so far, detailed
insights into the binding modes of known ATM inhibitors have been hampered due
to the lack of high-resolution ATM structures. Using cryo-EM, we have determined
the structure of human ATM to an overall resolution sufficient to build a
near-complete atomic model and identify two hitherto unknown zinc-binding
motifs. We determined the structure of the kinase domain bound to ATPγS and to
the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution,
respectively. The mode of action and selectivity of the ATM inhibitors can be
explained by structural comparison and provide a framework for structure-based
drug design.
Utilization of Metabolite Identification and Structural Data to Guide Design of Low-Dose IDO1 Inhibitors
Author: Hopkins B, Zhang H, Bharathan I, Li D, Pu Q, Zhou H, Martinot TA, Fradera X, Lammens A, Lesburg CA, Cohen RD, Ballard J, Knemeyer I, Otte K, Vincent S, Miller JR, Solban N, Cheng M, Geda P, Smotrov N, Song X, Bennett DJ, Han Y.
Publication with: Merck & Co.
Citation: ACS Med Chem Lett. 2021 Aug 18;12(9):1435-1440. doi: 10.1021/acsmedchemlett.1c00265. eCollection 2021 Sep 9.
Abstract:
Herein the discovery of potent IDO1 inhibitors with low predicted human dose is
discussed. Metabolite identification (MetID) and structural data were used to
strategically incorporate cyclopropane rings into this tetrahydronaphthyridine
series of IDO1 inhibitors to improve their metabolic stability and potency.
Enabling synthetic chemistry was developed to construct these unique fused
cyclopropyl compounds, leading to inhibitors with improved pharmacokinetics and
human whole blood potency and a predicted human oral dose as low as 9 mg once
daily (QD).
Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro
Author: Steinritz D, Lüling R, Siegert M, Mückter H, Popp T, Reinemer P, Gudermann T, Thiermann H, John H.
Publication with: Ludwig-Maximilians-Universität
Citation: Arch Toxicol. 2021 Oct;95(10):3253-3261. doi: 10.1007/s00204-021-03137-6. Epub 2021 Aug 16.
Abstract:
Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine
triphosphate (ATP) and creatine. The highly reactive free cysteine residue in
the active site of the enzyme (Cys283) is considered essential for the enzymatic
activity. In previous studies we demonstrated that Cys283 is targeted by the
alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with
a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on
rabbit muscle CK (rmCK) activity was investigated with special focus on the
alkylation of Cys283 and of reactive methionine (Met) residues. For
investigation of SM-alkylated amino acids in rmCK, micro liquid
chromatography-electrospray ionization high-resolution tandem-mass spectrometry
measurements were performed using the Orbitrap technology. The treatment of rmCK
with SM resulted in a decrease of enzyme activity. However, this decrease did
only weakly correlate to the modification of Cys283 but was conclusive for the
formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of
rmCK produced by side-directed mutagenesis that contained substitutions of the
respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly
resistant against SM. Our results point to a critical role of the surface
exposed Met70 and Met179 residues for CK activity.
Discovery of IACS-9779 and IACS-70465 as Potent Inhibitors Targeting Indoleamine 2,3-Dioxygenase 1 (IDO1) Apoenzyme
Author: Hamilton MM, Mseeh F, McAfoos TJ, Leonard PG, Reyna NJ, Harris AL, Xu A, Han M, Soth MJ, Czako B, Theroff JP, Mandal PK, Burke JP, Virgin-Downey B, Petrocchi A, Pfaffinger D, Rogers NE, Parker CA, Yu SS, Jiang Y, Krapp S, Lammens A, Trevitt G, Tremblay MR, Mikule K, Wilcoxen K, Cross JB, Jones P, Marszalek JR, Lewis RT.
Publication with: Tesaro Inc.
Citation: J Med Chem. 2021 Aug 12;64(15):11302-11329. doi: 10.1021/acs.jmedchem.1c00679. Epub 2021 Jul 22.
Abstract:
Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme that mediates the
rate-limiting step in the metabolism of l-tryptophan to kynurenine, has been
widely explored as a potential immunotherapeutic target in oncology. We
developed a class of inhibitors with a conformationally constrained
bicyclo[3.1.0]hexane core. These potently inhibited IDO1 in a cellular context
by binding to the apoenzyme, as elucidated by biochemical characterization and
X-ray crystallography. A SKOV3 tumor model was instrumental in differentiating
compounds, leading to the identification of IACS-9779 (62) and IACS-70465 (71).
IACS-70465 has excellent cellular potency, a robust pharmacodynamic response,
and in a human whole blood assay was more potent than linrodostat (BMS-986205).
IACS-9779 with a predicted human efficacious once daily dose below 1 mg/kg to
sustain >90% inhibition of IDO1 displayed an acceptable safety margin in rodent
toxicology and dog cardiovascular studies to support advancement into
preclinical safety evaluation for human development.
Aerosol-based ligand soaking of reservoir-free protein crystals
Author: Ross B, Krapp S, Geiss-Friedlander R, Littmann W, Huber R, Kiefersauer R.
Publication with: ATHENA Technologie Beratung GmbH
Citation: J Appl Crystallogr. 2021 May 28;54(Pt 3):895-902. doi: 10.1107/S1600576721003551. eCollection 2021 Jun 1.
Abstract:
Soaking of macromolecular crystals allows the formation of complexes via
diffusion of molecules into a preformed crystal for structural analysis. Soaking
offers various advantages over co-crystallization, e.g. small samples and
high-throughput experimentation. However, this method has disadvantages, such as
inducing mechanical stress on crystals and reduced success rate caused by low
affinity/solubility of the ligand. To bypass these issues, the Picodropper was
previously developed in the authors' laboratory. This technique aimed to deliver
small volumes of compound solution in response to crystal dehydration supported
by the Free Mounting System humidity control or by IR-laser-induced protein
crystal transformation. Herein, a new related soaking development, the
Aerosol-Generator, is introduced. This device delivers compounds onto the
solution-free surface of protein crystals using an ultrasonic technique. The
produced aerosol stream enables an easier and more accurate control of solution
volumes, reduced crystal handling, and crystal-size-independent soaking. The
Aerosol-Generator has been used to produce complexes of DPP8 crystals, where
otherwise regular soaking did not achieve complex formation. These results
demonstrate the potential of this device in challenging ligand-binding scenarios
and contribute to further understanding of DPP8 inhibitor binding.
Novel irreversible covalent BTK inhibitors discovered using DNA-encoded chemistry
Author: Guilinger JP, Archna A, Augustin M, Bergmann A, Centrella PA, Clark MA, Cuozzo JW, Däther M, Guié MA, Habeshian S, Kiefersauer R, Krapp S, Lammens A, Lercher L, Liu J, Liu Y, Maskos K, Mrosek M, Pflügler K, Siegert M, Thomson HA, Tian X, Zhang Y, Konz Makino DL, Keefe AD.
Publication with: X-Chem Inc.
Citation: Bioorg Med Chem. 2021 Jul 15;42:116223. doi: 10.1016/j.bmc.2021.116223. Epub 2021 May 19.
Abstract:
Libraries of DNA-Encoded small molecules created using combinatorial chemistry
and synthetic oligonucleotides are being applied to drug discovery projects
across the pharmaceutical industry. The majority of reported projects describe
the discovery of reversible, i.e. non-covalent, target modulators. We
synthesized multiple DNA-encoded chemical libraries terminated in electrophiles
and then used them to discover covalent irreversible inhibitors and report the
successful discovery of acrylamide- and epoxide-terminated Bruton's Tyrosine
Kinase (BTK) inhibitors. We also demonstrate their selectivity, potency and
covalent cysteine engagement using a range of techniques including X-ray
crystallography, thermal transition shift assay, reporter displacement assay and
intact protein complex mass spectrometry. The epoxide BTK inhibitors described
here are the first ever reported to utilize this electrophile for this target.
Discovery of a new series of PI3K-δ inhibitors from Virtual Screening
Author: Fradera X, Deng Q, Achab A, Garcia Y, Kattar SD, McGowan MA, Methot JL, Wilson K, Zhou H, Shaffer L, Goldenblatt P, Tong V, Augustin MA, Altman MD, Lesburg CA, Shah S, Katz JD.
Publication with: Merck & Co., Inc.
Citation: Bioorg Med Chem Lett. 2021 Jun 15;42:128046. doi: 10.1016/j.bmcl.2021.128046. Epub 2021 Apr 16.
Abstract:
PI3K-δ mediates key immune cell signaling pathways and is a target of interest for treatment of oncological and immunological disorders. Here we describe the discovery and optimization of a novel series of PI3K-δ selective inhibitors. We first identified hits containing an isoindolinone scaffold using a combined
ligand- and receptor-based virtual screening workflow, and then improved potency and selectivity guided by structural data and modeling. Careful optimization of
molecular properties led to compounds with improved permeability and pharmacokinetic profile, and high potency in a whole blood assay.
Carbamate and N-Pyrimidine Mitigate Amide Hydrolysis: Structure-Based Drug Design of Tetrahydroquinoline IDO1 Inhibitors
Author: Li D, Deng Y, Achab A, Bharathan I, Hopkins BA, Yu W, Zhang H, Sanyal S, Pu Q, Zhou H, Liu K, Lim J, Fradera X, Lesburg CA, Lammens A, Martinot TA, Cohen RD, Doty AC, Ferguson H, Nickbarg EB, Cheng M, Spacciapoli P, Geda P, Song X, Smotrov N, Abeywickrema P, Andrews C, Chamberlin C, Mabrouk O, Curran P, Richards M, Saradjian P, Miller JR, Knemeyer I, Otte KM, Vincent S, Sciammetta N, Pasternak A, Bennett DJ, Han Y.
Publication with: Merck & Co.
Citation: ACS Med Chem Lett. 2021 Feb 26;12(3):389-396. doi: 10.1021/acsmedchemlett.0c00525. eCollection 2021 Mar 11.
Abstract:
Indoleamine-2,3-dioxygenase-1 (IDO1) has emerged as an attractive target for
cancer immunotherapy. An automated ligand identification system screen afforded
the tetrahydroquinoline class of novel IDO1 inhibitors. Potency and
pharmacokinetic (PK) were key issues with this class of compounds.
Structure-based drug design and strategic incorporation of polarity enabled the
rapid improvement on potency, solubility, and oxidative metabolic stability.
Metabolite identification studies revealed that amide hydrolysis in the D-pocket
was the key clearance mechanism for this class. Strategic survey of amide
isosteres revealed that carbamates and N-pyrimidines, which maintained exquisite
potencies, mitigated the amide hydrolysis issue and led to an improved rat PK
profile. The lead compound 28 is a potent IDO1 inhibitor, with clean off-target
profiles and the potential for quaque die dosing in humans.
Structural basis for the regulation of nucleosome recognition and HDAC activity by histone deacetylase assemblies
Author: Lee JH, Bollschweiler D, Schäfer T, Huber R.
Publication with: Max Planck Institute of Biochemistry
Citation: Sci Adv. 2021 Jan 8;7(2):eabd4413. doi: 10.1126/sciadv.abd4413. Print 2021 Jan.
Abstract:
The chromatin-modifying histone deacetylases (HDACs) remove acetyl groups from
acetyl-lysine residues in histone amino-terminal tails, thereby mediating
transcriptional repression. Structural makeup and mechanisms by which
multisubunit HDAC complexes recognize nucleosomes remain elusive. Our
cryo-electron microscopy structures of the yeast class II HDAC ensembles show
that the HDAC protomer comprises a triangle-shaped assembly of stoichiometry
Hda12-Hda2-Hda3, in which the active sites of the Hda1 dimer are freely
accessible. We also observe a tetramer of protomers, where the nucleosome
binding modules are inaccessible. Structural analysis of the nucleosome-bound
complexes indicates how positioning of Hda1 adjacent to histone H2B affords HDAC
catalysis. Moreover, it reveals how an intricate network of multiple contacts
between a dimer of protomers and the nucleosome creates a platform for expansion
of the HDAC activities. Our study provides comprehensive insight into the
structural plasticity of the HDAC complex and its functional mechanism of
chromatin modification.
Optimization of Versatile Oxindoles as Selective PI3Kδ Inhibitors
Author: Methot JL, Achab A, Christopher M, Zhou H, McGowan MA, Trotter BW, Fradera X, Lesburg CA, Goldenblatt P, Hill A, Chen D, Otte KM, Augustin M, Shah S, Katz JD.
Publication with: Merck & Co.
Citation: ACS Med Chem Lett. 2020 Nov 19;11(12):2461-2469. doi: 10.1021/acsmedchemlett.0c00441. eCollection 2020 Dec 10.
Abstract:
The 3,3-disubstituted oxindole moiety is a versatile and rigid
three-dimensionally shaped scaffold. When engineered with a purine hinge-binding
core, exceptionally selective PI3Kδ kinase inhibitors were discovered by
exploiting small differences in isoform selectivity pockets. Crystal structures
of early lead 2f bound to PI3Kδ and PI3Kα helped rationalize the high
selectivity observed with 2f. By attenuating the lypophilicity and metabolic
liabilities of an oxindole moiety, we improved the preclinical species PK and
solubility and reduced adenosine uptake activity. The excellent potency and
kinome selectivity of 7-azaoxindole 4d and spirooxindole 5d, together with a low
plasma clearance and good half-life in rat and dog, supported a low once-daily
predicted human dose.
Activation of the IRE1 RNase through remodeling of the kinase front pocket by ATP-competitive ligands
Author: Ferri E, Le Thomas A, Wallweber HA, Day ES, Walters BT, Kaufman SE, Braun MG, Clark KR, Beresini MH, Mortara K, Chen YA, Canter B, Phung W, Liu PS, Lammens A, Ashkenazi A, Rudolph J, Wang W.
Publication with: Genentech, Inc.
Citation: Nat Commun. 2020 Dec 14;11(1):6387. doi: 10.1038/s41467-020-19974-5.
Abstract:
Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded
Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a
sensory lumenal domain, and tandem kinase and endoribonuclease (RNase)
cytoplasmic domains. Excess unfolded proteins in the ER lumen induce
dimerization and oligomerization of IRE1, triggering kinase
trans-autophosphorylation and RNase activation. Known ATP-competitive
small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize
the active dimeric unit, accordingly inhibiting or stimulating RNase activity.
Previous allosteric RNase activators display poor selectivity and/or weak
cellular activity. In this study, we describe a class of ATP-competitive RNase
activators possessing high selectivity and strong cellular activity. This class
of activators binds IRE1 in the kinase front pocket, leading to a distinct
conformation of the activation loop. Our findings reveal exquisitely precise
interdomain regulation within IRE1, advancing the mechanistic understanding of
this important enzyme and its investigation as a potential small-molecule
therapeutic target.
Optimization of linear and cyclic peptide inhibitors of KEAP1-NRF2 protein-protein interaction
Author: Colarusso S, De Simone D, Frattarelli T, Andreini M, Cerretani M, Missineo A, Moretti D, Tambone S, Kempf G, Augustin M, Steinbacher S, Munoz-Sanjuan I, Park L, Summa V, Tomei L, Bresciani A, Dominguez C, Toledo-Sherman L, Bianchi E.
Publication with: IRBM Spa
Citation: Bioorg Med Chem. 2020 Nov 1;28(21):115738. doi: 10.1016/j.bmc.2020.115738. Epub 2020 Aug 30.
Abstract:
Inhibition of KEAP1-NRF2 protein-protein interaction is considered a promising
strategy to selectively and effectively activate NRF2, a transcription factor
which is involved in several pathologies such as Huntington's disease (HD). A
library of linear peptides based on the NRF2-binding motifs was generated on the
nonapeptide lead Ac-LDEETGEFL-NH2 spanning residues 76-84 of the Neh2 domain of
NRF2 with the aim to replace E78, E79 and E82 with non-acidic amino acids. A
deeper understanding of the features and accessibility of the T80 subpocket was
also targeted by structure-based design. Approaches to improve cell permeability
were investigated using both different classes of cyclic peptides and
conjugation to cell-penetrating peptides. This insight will guide future design
of macrocycles, peptido-mimetics and, most importantly, small neutral
brain-penetrating molecules to evaluate whether NRF2 activators have utility in
HD.
A Single Second Shell Amino Acid Determines Affinity and Kinetics of Linagliptin Binding to Type 4 Dipeptidyl Peptidase and Fibroblast Activation Protein
Author: Schnapp G, Hoevels Y, Bakker RA, Schreiner P, Klein T, Nar H.
Publication with: Boehringer Ingelheim Pharma GmbH & Co. KG
Citation: ChemMedChem. 2021 Feb 17;16(4):630-639. doi: 10.1002/cmdc.202000591. Epub 2020 Oct 21.
Abstract:
Drugs targeting type 4 dipeptidyl peptidase (DPP-4) are beneficial for glycemic
control, whereas fibroblast activation protein alpha (FAP-α) is a potential
target for cancer therapies. Unlike other gliptins, linagliptin displays FAP
inhibition. We compared biophysical and structural characteristics of
linagliptin binding to DPP-4 and FAP to better understand what differentiates
linagliptin from other gliptins. Linagliptin exhibited high binding affinity (KD
) and a slow off-rate (koff ) when dissociating from DPP-4 (KD 6.6 pM; koff
5.1×10-5 s-1 ), and weaker inhibitory potency to FAP (KD 301 nM; koff >1 s-1 ).
Co-structures of linagliptin with DPP-4 or FAP were similar except for one
second shell amino acid difference: Asp663 (DPP-4) and Ala657 (FAP). pH
dependence of enzymatic activities and binding of linagliptin for DPP-4 and FAP
are dependent on this single amino acid difference. While linagliptin may not
display any anticancer activity at therapeutic doses, our findings may guide
future studies for the development of optimized inhibitors.
Discovery of Potent and Orally Available Bicyclo[1.1.1]pentane-Derived Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitors
Author: Pu Q, Zhang H, Guo L, Cheng M, Doty AC, Ferguson H, Fradera X, Lesburg CA, McGowan MA, Miller JR, Geda P, Song X, Otte K, Sciammetta N, Solban N, Yu W, Sloman DL, Zhou H, Lammens A, Neumann L, Bennett DJ, Pasternak A, Han Y.
Publication with: Merck & Co.
Citation: ACS Med Chem Lett. 2020 Jul 15;11(8):1548-1554. doi: 10.1021/acsmedchemlett.0c00195. eCollection 2020 Aug 13.
Abstract:
Indoleamine-2,3-dioxygenase 1 (IDO1) inhibition and its combination with immune
checkpoint inhibitors like pembrolizumab have drawn considerable attention from
both academia and the pharmaceutical industry. Here, we describe the discovery
of a novel class of highly potent IDO1 heme-displacing inhibitors featuring a
unique bicyclo[1.1.1]pentane motif. Compound 1, evolving from an ALIS (automated
ligand identification system) hit, exhibited excellent potency but lacked the
desired pharmacokinetic profile due to extensive amide hydrolysis of the
benzamide moiety. Replacing the central phenyl ring in 1 with a
bicyclo[1.1.1]pentane bioisostere effectively circumvented the amide hydrolysis
issue, resulting in the discovery of compound 2 with a favorable overall profile
such as excellent potency, selectivity, pharmacokinetics, and a low predicted
human dose.
Corrigendum to "Design of selective PI3Kδ inhibitors using an iterative scaffold-hopping workflow" [Bioorg. Med. Chem. Lett. 29 (2019) 2575-2580]
Author: Fradera X, Methot JL, Achab A, Christopher M, Altman MD, Zhou H, McGowan MA, Kattar SD, Wilson K, Garcia Y, Augustin MA, Lesburg CA, Shah S, Goldenblatt P, Katz JD.
Publication with: Merck & Co.
Citation: Bioorg Med Chem Lett. 2020 Aug 15;30(16):127363. doi: 10.1016/j.bmcl.2020.127363. Epub 2020 Jun 23.
Abstract:
Combined Peptide and Small-Molecule Approach toward Nonacidic THIQ Inhibitors of the KEAP1/NRF2 Interaction
Author: Ontoria JM, Biancofiore I, Fezzardi P, Ferrigno F, Torrente E, Colarusso S, Bianchi E, Andreini M, Patsilinakos A, Kempf G, Augustin M, Steinbacher S, Summa V, Pacifici R, Muñoz-Sanjuan I, Park L, Bresciani A, Dominguez C, Sherman LT, Harper S.
Publication with: IRBM S.p.A.
Citation: ACS Med Chem Lett. 2020 Apr 3;11(5):740-746. doi: 10.1021/acsmedchemlett.9b00594. eCollection 2020 May 14.
Abstract:
The NRF2-ARE pathway is an intrinsic mechanism of defense against oxidative
stress. Inhibition of the interaction between NRF2 and its main negative
regulator KEAP1 is an attractive strategy toward neuroprotective agents. We
report here the identification of nonacidic tetrahydroisoquinolines (THIQs) that
inhibit the KEAP1/NRF2 protein-protein interaction. Peptide SAR at one residue
is utilized as a tool to probe structural changes within a specific pocket of
the KEAP1 binding site. We used structural information from peptide screening at
the P2 pocket, noncovalent small-molecules inhibitors, and the outcome from an
explorative SAR at position 5 of THIQs to identify a series of neutral THIQ
analogs that bind to KEAP1 in the low micromolar range. These analogs establish
new H-bond interactions at the P3 and P2 pockets allowing the replacement of the
carboxylic acid functionality by a neutral primary carboxamide. X-ray
crystallographic studies reveal the novel binding mode of these molecules to
KEAP1.
Strategic Incorporation of Polarity in Heme-Displacing Inhibitors of Indoleamine-2,3-dioxygenase-1 (IDO1)
Author: White C, McGowan MA, Zhou H, Sciammetta N, Fradera X, Lim J, Joshi EM, Andrews C, Nickbarg EB, Cowley P, Trewick S, Augustin M, von Köenig K, Lesburg CA, Otte K, Knemeyer I, Woo H, Yu W, Cheng M, Spacciapoli P, Geda P, Song X, Smotrov N, Curran P, Heo MR, Abeywickrema P, Miller JR, Bennett DJ, Han Y.
Publication with: Merck & Co.
Citation: ACS Med Chem Lett. 2020 Mar 10;11(4):550-557. doi: 10.1021/acsmedchemlett.0c00010. eCollection 2020 Apr 9.
Abstract:
Indoleamine-2,3-dioxygenase-1 (IDO1) has emerged as a target of significant
interest to the field of cancer immunotherapy, as the upregulation of IDO1 in
certain cancers has been linked to host immune evasion and poor prognosis for
patients. In particular, IDO1 inhibition is of interest as a combination therapy
with immune checkpoint inhibition. Through an Automated Ligand Identification
System (ALIS) screen, a diamide class of compounds was identified as a promising
lead for the inhibition of IDO1. While hit 1 possessed attractive cell-based
potency, it suffered from a significant right-shift in a whole blood assay, poor
solubility, and poor pharmacokinetic properties. Through a physicochemical
property-based approach, including a focus on lowering AlogP98 via the strategic
introduction of polar substitution, compound 13 was identified bearing a pyridyl
oxetane core. Compound 13 demonstrated improved whole blood potency and
solubility, and an improved pharmacokinetic profile resulting in a low predicted
human dose.
Discovery of sulfonamides and 9-oxo-2,8-diazaspiro[5,5]undecane-2-carboxamides as human kynurenine aminotransferase 2 (KAT2) inhibitors
Author: Kalliokoski T, Rummakko P, Rantanen M, Blaesse M, Augustin M, Ummenthala GR, Choudhary S, Venäläinen J.
Publication with: Orion Pharma
Citation: Bioorg Med Chem Lett. 2020 Apr 15;30(8):127060. doi: 10.1016/j.bmcl.2020.127060. Epub 2020 Feb 22.
Abstract:
Human kynurenine aminotransferase 2 (KAT2) inhibitors could be potentially used
to treat the cognitive deficits associated with bipolar disease and
schizophrenia. Although, there has been active drug research activity by several
industrial and academic groups in developing KAT2 inhibitors over the years, no
such compound has proceeded to the clinics. Here, we report two different
chemical series of reversible KAT2 inhibitors with sub-micromolar activities.
The first series was identified by a high-throughput screening of a diverse
random library and the second one by structure-based virtual screening. Two
novel crystal structures of KAT2 complexed with different reversible inhibitors
were also deposited to the Protein databank which could be useful for future
drug discovery efforts.
A precisely positioned MED12 activation helix stimulates CDK8 kinase activity
Author: Klatt F, Leitner A, Kim IV, Ho-Xuan H, Schneider EV, Langhammer F, Weinmann R, Müller MR, Huber R, Meister G, Kuhn CD.
Publication with: Max Planck Institute of Biochemistry
Citation: Proc Natl Acad Sci U S A. 2020 Feb 11;117(6):2894-2905. doi: 10.1073/pnas.1917635117. Epub 2020 Jan 27.
Abstract:
The Mediator kinase module regulates eukaryotic transcription by phosphorylating
transcription-related targets and by modulating the association of Mediator and
RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8
(CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its
deregulation contributing to numerous malignancies. Here, we combine in vitro
biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to
describe the binding location of the N-terminal segment of MED12 on the
CDK8/Cyclin C complex and to gain mechanistic insights into the activation of
CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps
around CDK8, whereby it positions an "activation helix" close to the T-loop of
CDK8 for its activation. Intriguingly, mutations in the activation helix that
are frequently found in cancers do not diminish the affinity of MED12 for CDK8,
yet likely alter the exact positioning of the activation helix. Furthermore, we
find the transcriptome-wide gene-expression changes in human cells that result
from a mutation in the MED12 activation helix to correlate with deregulated
genes in breast and colon cancer. Finally, functional assays in the presence of
kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8
and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase
inhibitors. Taken together, our results not only allow us to propose a revised
model of how CDK8 activity is regulated by MED12, but also offer a path forward
in developing small molecules that target CDK8 in its MED12-bound form.
Discovery and optimization of heteroaryl piperazines as potent and selective PI3Kδ inhibitors
Author: Zhou H, McGowan MA, Lipford K, Christopher M, Fradera X, Witter D, Lesburg CA, Li C, Methot JL, Lampe J, Achab A, Shaffer L, Goldenblatt P, Shah S, Bass A, Schroeder G, Chen D, Zeng H, Augustin MA, Katz JD.
Publication with: Merck & Co.
Citation: Bioorg Med Chem Lett. 2020 Jan 1;30(1):126715. doi: 10.1016/j.bmcl.2019.126715. Epub 2019 Oct 18.
Abstract:
A high-throughput screening (HTS) campaign identified a class of heteroaryl
piperazines with excellent baseline affinity and selectivity for
phosphoinositide 3-kinase δ (PI3Kδ) over closely related isoforms. Rapid
evaluation and optimization of structure-activity relationships (SAR) for this
class, leveraging the modular nature of this scaffold, facilitated development
of this hit class into a series of potent and selective inhibitors of PI3Kδ.
This effort culminated in the identification of 29, which displayed excellent
potency in enzyme and cell-based assays, as well as favorable pharmacokinetic
and off-target profiles.
Discovery of novel Cyclophilin D inhibitors starting from three dimensional fragments with millimolar potencies
Author: Grädler U, Schwarz D, Blaesse M, Leuthner B, Johnson TL, Bernard F, Jiang X, Marx A, Gilardone M, Lemoine H, Roche D, Jorand-Lebrun C.
Publication with: Merck KGaA
Citation: Bioorg Med Chem Lett. 2019 Dec 1;29(23):126717. doi: 10.1016/j.bmcl.2019.126717. Epub 2019 Oct 16.
Abstract:
Fragment-based screening by SPR enabled the discovery of chemical diverse
fragment hits with millimolar binding affinities to the peptidyl-prolyl
isomerase Cyclophilin D (CypD). The CypD protein crystal structures of 6
fragment hits provided the basis for subsequent medicinal chemistry optimization
by fragment merging and linking yielding three different chemical series with
either urea, oxalyl or amide linkers connecting millimolar fragments in the S1'
and S2 pockets. We successfully improved the in vitro CypD potencies in the
biochemical FP and PPIase assays and in the biophysical SPR binding assay from
millimolar towards the low micromolar and submicromolar range by >1000-fold for
some fragment derivatives. The initial SAR together with the protein crystal
structures of our novel CypD inhibitors provide a suitable basis for further
hit-to-lead optimization.
Design of selective PI3Kδ inhibitors using an iterative scaffold-hopping workflow
Author: Fradera X, Methot JL, Achab A, Christopher M, Altman MD, Zhou H, McGowan MA, Kattar SD, Wilson K, Garcia Y, Augustin MA, Lesburg CA, Shah S, Goldenblatt P, Katz JD.
Publication with: Merck & Co.
Citation: Bioorg Med Chem Lett. 2019 Sep 15;29(18):2575-2580. doi: 10.1016/j.bmcl.2019.08.004. Epub 2019 Aug 5.
Abstract:
PI3Kδ mediates key immune cell signaling pathways and is a target of interest
for multiple indications in immunology and oncology. Here we report a
structure-based scaffold-hopping strategy for the design of chemically diverse
PI3Kδ inhibitors. Using this strategy, we identified several scaffolds that can
be combined to generate new PI3Kδ inhibitors with high potency and isoform
selectivity. In particular, an oxindole-based scaffold was found to impart
exquisite selectivity when combined with several hinge binding motifs.
Design and Evaluation of Highly Selective Human Immunoproteasome Inhibitors Reveal a Compensatory Process That Preserves Immune Cell Viability
Author: Ladi E, Everett C, Stivala CE, Daniels BE, Durk MR, Harris SF, Huestis MP, Purkey HE, Staben ST, Augustin M, Blaesse M, Steinbacher S, Eidenschenk C, Pappu R, Siu M.
Publication with: Proteros Biostructures GmbH
Citation: J Med Chem. 2019 Aug 8;62(15):7032-7041. doi: 10.1021/acs.jmedchem.9b00509. Epub 2019 Jul 29.
Abstract:
The pan-proteasome inhibitor bortezomib demonstrated clinical efficacy in
off-label trials of Systemic Lupus Erythematosus. One potential mechanism of
this clinical benefit is from the depletion of pathogenic immune cells
(plasmablasts and plasmacytoid dendritic cells). However, bortezomib is
cytotoxic against nonimmune cells, which limits its use for autoimmune diseases.
An attractive alternative is to selectively inhibit the immune cell-specific
immunoproteasome to deplete pathogenic immune cells and spare nonhematopoietic
cells. Here, we disclose the development of highly subunit-selective
immunoproteasome inhibitors using insights obtained from the first bona fide
human immunoproteasome cocrystal structures. Evaluation of these inhibitors
revealed that immunoproteasome-specific inhibition does not lead to immune cell
death as anticipated and that targeting viability requires inhibition of both
immuno- and constitutive proteasomes. CRISPR/Cas9-mediated knockout experiments
confirmed upregulation of the constitutive proteasome upon disruption of the
immunoproteasome, protecting cells from death. Thus, immunoproteasome inhibition
alone is not a suitable approach to deplete immune cells.
Total Synthesis of Covalent Cysteine Protease Inhibitor N-Desmethyl Thalassospiramide C and Crystallographic Evidence for Its Mode of Action
Author: Fournier J, Chen K, Mailyan AK, Jackson JJ, Buckman BO, Emayan K, Yuan S, Rajagopalan R, Misialek S, Adler M, Blaesse M, Griessner A, Zakarian A.
Publication with: Blade Therapeutics
Citation: Org Lett. 2019 Jan 18;21(2):508-512. doi: 10.1021/acs.orglett.8b03821. Epub 2019 Jan 10.
Abstract:
A total synthesis of N-desmethyl thalassospiramide C, a unique strained
macrocyclic proteobacterial depsipeptide, enabled a detailed crystallographic
study of its covalent complex with cathepsin K, a member of a medicinally
important family of cysteine proteases. The study provides support for the
mechanism of action, and the insight gained can be used for structure-based drug
design targeting these calpain proteases.
Enhancement of therapeutic potential of a naturally occurring human antibody targeting a phosphorylated Ser(422) containing epitope on pathological tau
Author: van Ameijde J, Crespo R, Janson R, Juraszek J, Siregar B, Verveen H, Sprengers I, Nahar T, Hoozemans JJ, Steinbacher S, Willems R, Delbroek L, Borgers M, Dockx K, Van Kolen K, Mercken M, Pascual G, Koudstaal W, Apetri A.
Publication with: Janssen Pharmaceutical Companies of Johnson and Johnson
Citation: Acta Neuropathol Commun. 2018 Jul 12;6(1):59. doi: 10.1186/s40478-018-0562-9.
Abstract:
Aggregation of tau protein and spreading of tau aggregates are pivotal
pathological processes in a range of neurological disorders. Accumulating
evidence suggests that immunotherapy targeting tau may be a viable therapeutic
strategy. We have previously described the isolation of antibody CBTAU-22.1 from
the memory B-cell repertoire of healthy human donors. CBTAU-22.1 was shown to
specifically bind a disease-associated phosphorylated epitope in the C-terminus
of tau (Ser422) and to be able to inhibit the spreading of pathological tau
aggregates from P301S spinal cord lysates in vitro, albeit with limited potency.
Using a combination of rational design and random mutagenesis we have derived a
variant antibody with improved affinity while maintaining the specificity of the
parental antibody. This affinity improved antibody showed greatly enhanced
potency in a cell-based immunodepletion assay using paired helical filaments
(PHFs) derived from human Alzheimer's disease (AD) brain tissue. Moreover, the
affinity improved antibody limits the in vitro aggregation propensity of full
length tau species specifically phosphorylated at position 422 produced by
employing a native chemical ligation approach. Together, these results indicate
that in addition to being able to inhibit the spreading of pathological tau
aggregates, the matured antibody can potentially also interfere with the
nucleation of tau which is believed to be the first step of the pathogenic
process. Finally, the functionality in a P301L transgenic mice co-injection
model highlights the therapeutic potential of human antibody dmCBTAU-22.1.
Remodeling and Repositioning of Nucleosomes in Nucleosomal Arrays
Author: Ludwigsen J, Hepp N, Klinker H, Pfennig S, Mueller-Planitz F.
Publication with: LMU Munich
Citation: Methods Mol Biol. 2018;1805:349-370. doi: 10.1007/978-1-4939-8556-2_18.
Abstract:
ATP-dependent nucleosome remodeling factors sculpt the nucleosomal landscape of
eukaryotic chromatin. They deposit or evict nucleosomes or reposition them along
DNA in a process termed nucleosome sliding. Remodeling has traditionally been
analyzed using mononucleosomes as a model substrate. In vivo, however,
nucleosomes form extended arrays with regular spacing. Here, we describe how
regularly spaced nucleosome arrays can be reconstituted in vitro and how these
arrays can be used to dissect remodeling in the test tube. We outline two
assays. The first assay senses various structural changes to a specific
nucleosome within the nucleosomal array whereas the second assay is specific
toward detecting repositioning of nucleosomes within the array. Both assays
exploit changes to the accessibility of DNA to restriction enzymes during the
remodeling reaction.
Discovery of a Potent, Orally Bioavailable PI4KIIIβ Inhibitor (UCB9608) Able To Significantly Prolong Allogeneic Organ Engraftment in Vivo
Author: Reuberson J, Horsley H, Franklin RJ, Ford D, Neuss J, Brookings D, Huang Q, Vanderhoydonck B, Gao LJ, Jang MY, Herdewijn P, Ghawalkar A, Fallah-Arani F, Khan AR, Henshall J, Jairaj M, Malcolm S, Ward E, Shuttleworth L, Lin Y, Li S, Louat T, Waer M, Herman J, Payne A, Ceska T, Doyle C, Pitt W, Calmiano M, Augustin M, Steinbacher S, Lammens A, Allen R.
Publication with: UCB Pharma
Citation: J Med Chem. 2018 Aug 9;61(15):6705-6723. doi: 10.1021/acs.jmedchem.8b00521. Epub 2018 Jul 19.
Abstract:
The primary target of a novel series of immunosuppressive
7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid
kinase, PI4KIIIβ. Evaluation of the series highlighted their poor solubility and
unwanted off-target activities. A medicinal chemistry strategy was put in place
to optimize physicochemical properties within the series, while maintaining
potency and improving selectivity over other lipid kinases. Compound 22 was
initially identified and profiled in vivo, before further modifications led to
the discovery of 44 (UCB9608), a vastly more soluble, selective compound with
improved metabolic stability and excellent pharmacokinetic profile. A co-crystal
structure of 44 with PI4KIIIβ was solved, confirming the binding mode of this
class of inhibitor. The much-improved in vivo profile of 44 positions it as an
ideal tool compound to further establish the link between PI4KIIIβ inhibition
and prolonged allogeneic organ engraftment, and suppression of immune responses
in vivo.
A common antigenic motif recognized by naturally occurring human V(H)5-51/V(L)4-1 anti-tau antibodies with distinct functionalities
Author: Apetri A, Crespo R, Juraszek J, Pascual G, Janson R, Zhu X, Zhang H, Keogh E, Holland T, Wadia J, Verveen H, Siregar B, Mrosek M, Taggenbrock R, Ameijde J, Inganäs H, van Winsen M, Koldijk MH, Zuijdgeest D, Borgers M, Dockx K, Stoop EJM, Yu W, Brinkman-van der Linden EC, Ummenthum K, van Kolen K, Mercken M, Steinbacher S, de Marco D, Hoozemans JJ, Wilson IA, Koudstaal W, Goudsmit J.
Publication with: Janssen Pharmaceutical Companies of Johnson & Johnson
Citation: Acta Neuropathol Commun. 2018 May 31;6(1):43. doi: 10.1186/s40478-018-0543-z.
Abstract:
Misfolding and aggregation of tau protein are closely associated with the onset
and progression of Alzheimer's Disease (AD). By interrogating IgG+ memory B
cells from asymptomatic donors with tau peptides, we have identified two
somatically mutated VH5-51/VL4-1 antibodies. One of these, CBTAU-27.1, binds to
the aggregation motif in the R3 repeat domain and blocks the aggregation of tau
into paired helical filaments (PHFs) by sequestering monomeric tau. The other,
CBTAU-28.1, binds to the N-terminal insert region and inhibits the spreading of
tau seeds and mediates the uptake of tau aggregates into microglia by binding
PHFs. Crystal structures revealed that the combination of VH5-51 and VL4-1
recognizes a common Pro-Xn-Lys motif driven by germline-encoded hotspot
interactions while the specificity and thereby functionality of the antibodies
are defined by the CDR3 regions. Affinity improvement led to improvement in
functionality, identifying their epitopes as new targets for therapy and
prevention of AD.
β-Glucocerebrosidase Modulators Promote Dimerization of β-Glucocerebrosidase and Reveal an Allosteric Binding Site
Author: Zheng J, Chen L, Skinner OS, Ysselstein D, Remis J, Lansbury P, Skerlj R, Mrosek M, Heunisch U, Krapp S, Charrow J, Schwake M, Kelleher NL, Silverman RB, Krainc D.
Publication with: Northwestern University, Chicago
Citation: J Am Chem Soc. 2018 May 9;140(18):5914-5924. doi: 10.1021/jacs.7b13003. Epub 2018 Apr 30.
Abstract:
β-Glucocerebrosidase (GCase) mutations cause Gaucher's disease and are a high
risk factor in Parkinson's disease. The implementation of a small molecule
modulator is a strategy to restore proper folding and lysosome delivery of
degradation-prone mutant GCase. Here, we present a potent quinazoline modulator,
JZ-4109, which stabilizes wild-type and N370S mutant GCase and increases GCase
abundance in patient-derived fibroblast cells. We then developed a covalent
modification strategy using a lysine targeted inactivator (JZ-5029) for in vitro
mechanistic studies. By using native top-down mass spectrometry, we located two
potentially covalently modified lysines. We obtained the first crystal
structure, at 2.2 Å resolution, of a GCase with a noniminosugar modulator
covalently bound, and were able to identify the exact lysine residue modified
(Lys346) and reveal an allosteric binding site. GCase dimerization was induced
by our modulator binding, which was observed by native mass spectrometry, its
crystal structure, and size exclusion chromatography with a multiangle light
scattering detector. Finally, the dimer form was confirmed by negative staining
transmission electron microscopy studies. Our newly discovered allosteric site
and observed GCase dimerization provide a new mechanistic insight into GCase and
its noniminosugar modulators and facilitate the rational design of novel GCase
modulators for Gaucher's disease and Parkinson's disease.
A loop region of BAFF controls B cell survival and regulates recognition by different inhibitors
Author: Vigolo M, Chambers MG, Willen L, Chevalley D, Maskos K, Lammens A, Tardivel A, Das D, Kowalczyk-Quintas C, Schuepbach-Mallepell S, Smulski CR, Eslami M, Rolink A, Hummler E, Samy E, Fomekong Nanfack Y, Mackay F, Liao M, Hess H, Jiang X, Schneider P.
Publication with: Harvard Medical School
Citation: Nat Commun. 2018 Mar 23;9(1):1199. doi: 10.1038/s41467-018-03323-8.
Abstract:
The B cell survival factor (TNFSF13B/BAFF) is often elevated in autoimmune
diseases and is targeted in the clinic for the treatment of systemic lupus
erythematosus. BAFF contains a loop region designated the flap, which is
dispensable for receptor binding. Here we show that the flap of BAFF has two
functions. In addition to facilitating the formation of a highly active BAFF
60-mer as shown previously, it also converts binding of BAFF to TNFRSF13C
(BAFFR) into a signaling event via oligomerization of individual BAFF-BAFFR
complexes. Binding and activation of BAFFR can therefore be targeted
independently to inhibit or activate the function of BAFF. Moreover, structural
analyses suggest that the flap of BAFF 60-mer temporarily prevents binding of an
anti-BAFF antibody (belimumab) but not of a decoy receptor (atacicept). The
observed differences in profiles of BAFF inhibition may confer distinct
biological and clinical efficacies to these therapeutically relevant inhibitors.
Structures and mechanism of dipeptidyl peptidases 8 and 9, important players in cellular homeostasis and cancer
Author: Ross B, Krapp S, Augustin M, Kierfersauer R, Arciniega M, Geiss-Friedlander R, Huber R.
Publication with: Max-Planck-Institute for Biochemistry
Citation: Proc Natl Acad Sci U S A. 2018 Feb 13;115(7):E1437-E1445. doi: 10.1073/pnas.1717565115. Epub 2018 Jan 30.
Abstract:
Dipeptidyl peptidases 8 and 9 are intracellular N-terminal dipeptidyl peptidases
(preferentially postproline) associated with pathophysiological roles in immune
response and cancer biology. While the DPP family member DPP4 is extensively
characterized in molecular terms as a validated therapeutic target of type II
diabetes, experimental 3D structures and ligand-/substrate-binding modes of DPP8
and DPP9 have not been reported. In this study we describe crystal and molecular
structures of human DPP8 (2.5 Å) and DPP9 (3.0 Å) unliganded and complexed with
a noncanonical substrate and a small molecule inhibitor, respectively. Similar
to DPP4, DPP8 and DPP9 molecules consist of one β-propeller and α/β hydrolase
domain, forming a functional homodimer. However, they differ extensively in the
ligand binding site structure. In intriguing contrast to DPP4, where liganded
and unliganded forms are closely similar, ligand binding to DPP8/9 induces an
extensive rearrangement at the active site through a disorder-order transition
of a 26-residue loop segment, which partially folds into an α-helix (R-helix),
including R160/133, a key residue for substrate binding. As vestiges of this
helix are also seen in one of the copies of the unliganded form, conformational
selection may contributes to ligand binding. Molecular dynamics simulations
support increased flexibility of the R-helix in the unliganded state.
Consistently, enzyme kinetics assays reveal a cooperative allosteric mechanism.
DPP8 and DPP9 are closely similar and display few opportunities for targeted
ligand design. However, extensive differences from DPP4 provide multiple cues
for specific inhibitor design and development of the DPP family members as
therapeutic targets or antitargets.
Molecular Insights into Function and Competitive Inhibition of Pseudomonas aeruginosa Multiple Virulence Factor Regulator
Author: Kitao T, Lepine F, Babloudi S, Walte F, Steinbacher S, Maskos K, Blaesse M, Negri M, Pucci M, Zahler B, Felici A, Rahme LG.
Publication with: Harvard Medical School,
Citation: mBio. 2018 Jan 16;9(1):e02158-17. doi: 10.1128/mBio.02158-17.
Abstract:
New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by multidrug-resistant (MDR) bacteria. Multiple virulence factor regulator (MvfR or PqsR), a Pseudomonas aeruginosa quorum sensing transcription factor, regulates functions important in both acute and
persistent infections. Recently identified non-ligand-based
benzamine-benzimidazole (BB) inhibitors of MvfR suppress both acute and persistent P. aeruginosa infections in mice without perturbing bacterial growth.
Here, we elucidate the crystal structure of the MvfR ligand binding domain (LBD) in complex with one potent BB inhibitor, M64. Structural analysis indicated that M64 binds, like native ligands, to the MvfR hydrophobic cavity. A hydrogen bond
and pi interaction were found to be important for MvfR-M64 affinity. Surface plasmon resonance analysis demonstrated that M64 is a competitive inhibitor of MvfR. Moreover, a protein engineering approach revealed that Gln194 and Tyr258 are critical for the interaction between MvfR and M64. Random mutagenesis of the full-length MvfR protein identified a single-amino-acid substitution, I68F, at a DNA binding linker domain that confers M64 insensitivity. In the presence of
M64, I68F but not the wild-type (WT) MvfR protein retained DNA binding ability.
Our findings strongly suggest that M64 promotes conformational change at the DNA binding domain of MvfR and that the I68F mutation may compensate for this change, indicating allosteric inhibition. This work provides critical new insights into the molecular mechanism of MvfR function and inhibition that could aid in the optimization of anti-MvfR compounds and improve our understanding of
MvfRregulation.IMPORTANCEPseudomonas aeruginosa is an opportunistic
Gram-negative pathogen that causes serious acute, persistent, and relapsing infections. New approaches to antimicrobial drug discovery are urgently needed to combat intractable infections caused by this pathogen. The Pseudomonas aeruginosa quorum sensing transcription factor MvfR regulates functions important in both acute and persistent infections. We used recently identified inhibitors of MvfR to perform structural studies and reveal important insights that would benefit the optimization of anti-MvfR compounds. Altogether, the results reported here provide critical detailed mechanistic insights into the
function of MvfR domains that may benefit the optimization of the chemical,
pharmacological, and safety properties of MvfR antagonist series.
The Y. bercovieri Anbu crystal structure sheds light on the evolution of highly (pseudo)symmetric multimers
Author: Piasecka A, Czapinska H, Vielberg MT, Szczepanowski RH, Kiefersauer R, Reed S, Groll M, Bochtler M.
Publication with: Institute of Biochemistry and Biophysics, Warsaw, Poland
Citation: J Mol Biol. 2018 Mar 2;430(5):611-627. doi: 10.1016/j.jmb.2017.11.016. Epub 2017 Dec 16.
Abstract:
Ancestral β-subunit (Anbu) is homologous to HslV and 20S proteasomes. Based on
its phylogenetic distribution and sequence clustering, Anbu has been proposed as the "ancestral" form of proteasomes. Here, we report biochemical data, small-angle X-ray scattering results, negative-stain electron microscopy micrographs and a crystal structure of the Anbu particle from Yersinia bercovieri (YbAnbu). All data are consistent with YbAnbu forming defined 12-14 subunit multimers that differ in shape from both HslV and 20S proteasomes. The crystal structure reveals that YbAnbu subunits form tight dimers, held together in part by the Anbu specific C-terminal helices. These dimers ("protomers") further assemble into a low-rise left-handed staircase. The lock-washer shape of YbAnbu is consistent with the presence of defined multimers, X-ray diffraction data in solution and negative-stain electron microscopy images. The presented structure suggests a possible evolutionary pathway from helical filaments to highly symmetric or pseudosymmetric multimer structures. YbAnbu subunits have the Ntn-hydrolase fold, a putative S1 pocket and conserved candidate catalytic
residues Thr1, Asp17 and Lys32(33). Nevertheless, we did not detect any YbAnbu peptidase or amidase activity. However, we could document orthophosphate production from ATP catalyzed by the ATP-grasp protein encoded in the Y. bercovieri Anbu operon.
Discovery of Peptidomimetic Antibody-Drug Conjugate Linkers with Enhanced Protease Specificity
Author: Wei B, Gunzner-Toste J, Yao H, Wang T, Wang J, Xu Z, Chen J, Wai J, Nonomiya J, Tsai SP, Chuh J, Kozak KR, Liu Y, Yu SF, Lau J, Li G, Phillips GD, Leipold D, Kamath A, Su D, Xu K, Eigenbrot C, Steinbacher S, Ohri R, Raab H, Staben LR, Zhao G, Flygare JA, Pillow TH, Verma V, Masterson LA, Howard PW, Safina B.
Publication with: Genentech, Inc.
Citation: J Med Chem. 2018 Feb 8;61(3):989-1000. doi: 10.1021/acs.jmedchem.7b01430. Epub 2017 Dec 21.
Abstract:
Antibody-drug conjugates (ADCs) have become an important therapeutic modality
for oncology, with three approved by the FDA and over 60 others in clinical
trials. Despite the progress, improvements in ADC therapeutic index are desired.
Peptide-based ADC linkers that are cleaved by lysosomal proteases have shown
sufficient stability in serum and effective payload-release in targeted cells.
If the linker can be preferentially hydrolyzed by tumor-specific proteases,
safety margin may improve. However, the use of peptide-based linkers limits our
ability to modulate protease specificity. Here we report the structure-guided
discovery of novel, nonpeptidic ADC linkers. We show that a
cyclobutane-1,1-dicarboxamide-containing linker is hydrolyzed predominantly by
cathepsin B while the valine-citrulline dipeptide linker is not. ADCs bearing
the nonpeptidic linker are as efficacious and stable in vivo as those with the
dipeptide linker. Our results strongly support the application of the
peptidomimetic linker and present new opportunities for improving the
selectivity of ADCs.
His-FLAG Tag as a Fusion Partner of Glycosylated Human Interferon-Gamma and Its Mutant: Gain or Loss?
Author: Krachmarova E, Tileva M, Lilkova E, Petkov P, Maskos K, Ilieva N, Ivanov I, Litov L, Nacheva G.
Publication with: Sofia University, Bulgaria
Citation: Biomed Res Int. 2017;2017:3018608. doi: 10.1155/2017/3018608. Epub 2017 Jun 8.
Abstract:
In order to obtain glycosylated human interferon-gamma (hIFNγ) and its highly
prone to aggregation mutant K88Q, a secretory expression in insect cells was
employed. To facilitate recombinant proteins purification, detection, and
stability the baculovirus expression vectors were constructed to bear N-terminal
His6-FLAG tag. Although the obtained proteins were glycosylated, we found that
their biological activity was 100 times lower than expected. Our attempts to
recover the biological properties of both proteins by tag removal failed due to
enterokinase resistance of the tag. Surprisingly, the tag was easily cleaved
when the proteins were expressed in E. coli cells and the tag-free proteins
showed fully restored activity. To shed light on this phenomenon we performed
molecular dynamics simulations. The latter showed that the tags interact with
the receptor binding domains and the flexible C-termini of the fusion proteins
thus suppressing their complex formation with the hIFNγ receptor. We hypothesize
that in the case of glycosylated proteins the tag/C-terminal interaction
positions the FLAG peptide in close proximity to the glycans thus sterically
impeding the enterokinase access to its recognition site.
Discovery of Highly Potent and Selective Small-Molecule Reversible Factor D Inhibitors Demonstrating Alternative Complement Pathway Inhibition in Vivo
Author: Lorthiois E, Anderson K, Vulpetti A, Rogel O, Cumin F, Ostermann N, Steinbacher S, Mac Sweeney A, Delgado O, Liao SM, Randl S, Rüdisser S, Dussauge S, Fettis K, Kieffer L, de Erkenez A, Yang L, Hartwieg C, Argikar UA, La Bonte LR, Newton R, Kansara V, Flohr S, Hommel U, Jaffee B, Maibaum J.
Publication with: Novartis Pharma AG
Citation: J Med Chem. 2017 Jul 13;60(13):5717-5735. doi: 10.1021/acs.jmedchem.7b00425. Epub 2017 Jun 30.
Abstract:
The highly specific S1 serine protease factor D (FD) plays a central role in the
amplification of the complement alternative pathway (AP) of the innate immune
system. Genetic associations in humans have implicated AP activation in
age-related macular degeneration (AMD), and AP dysfunction predisposes
individuals to disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and
atypical hemolytic uremic syndrome (aHUS). The combination of structure-based
hit identification and subsequent optimization of the center (S)-proline-based
lead 7 has led to the discovery of noncovalent reversible and selective human
factor D (FD) inhibitors with drug-like properties. The orally bioavailable
compound 2 exerted excellent potency in 50% human whole blood in vitro and
blocked AP activity ex vivo after oral administration to monkeys as demonstrated
by inhibition of membrane attack complex (MAC) formation. Inhibitor 2
demonstrated sustained oral and ocular efficacy in a model of lipopolysaccharide
(LPS)-induced systemic AP activation in mice expressing human FD.
Discovery of Selective Phosphodiesterase 1 Inhibitors with Memory Enhancing Properties
Author: Dyck B, Branstetter B, Gharbaoui T, Hudson AR, Breitenbucher JG, Gomez L, Botrous I, Marrone T, Barido R, Allerston CK, Cedervall EP, Xu R, Sridhar V, Barker R, Aertgeerts K, Schmelzer K, Neul D, Lee D, Massari ME, Andersen CB, Sebring K, Zhou X, Petroski R, Limberis J, Augustin M, Chun LE, Edwards TE, Peters M, Tabatabaei A.
Publication with: Dart Neuroscience LLC
Citation: J Med Chem. 2017 Apr 27;60(8):3472-3483. doi: 10.1021/acs.jmedchem.7b00302. Epub 2017 Apr 13.
Abstract:
A series of potent thienotriazolopyrimidinone-based PDE1 inhibitors was
discovered. X-ray crystal structures of example compounds from this series in
complex with the catalytic domain of PDE1B and PDE10A were determined, allowing
optimization of PDE1B potency and PDE selectivity. Reduction of hERG affinity
led to greater than a 3000-fold selectivity for PDE1B over hERG.
6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydropyrido[4',3':4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one
was identified as an orally bioavailable and brain penetrating PDE1B enzyme
inhibitor with potent memory-enhancing effects in a rat model of object
recognition memory.
Structural Basis of Small-Molecule Aggregate Induced Inhibition of a Protein-Protein Interaction
Author: Blevitt JM, Hack MD, Herman KL, Jackson PF, Krawczuk PJ, Lebsack AD, Liu AX, Mirzadegan T, Nelen MI, Patrick AN, Steinbacher S, Milla ME, Lumb KJ.
Publication with: Janssen R&D LLC
Citation: J Med Chem. 2017 Apr 27;60(8):3511-3517. doi: 10.1021/acs.jmedchem.6b01836. Epub 2017 Mar 16.
Abstract:
A prevalent observation in high-throughput screening and drug discovery programs
is the inhibition of protein function by small-molecule compound aggregation.
Here, we present the X-ray structural description of aggregation-based
inhibition of a protein-protein interaction involving tumor necrosis factor α
(TNFα). An ordered conglomerate of an aggregating small-molecule inhibitor
(JNJ525) induces a quaternary structure switch of TNFα that inhibits the
protein-protein interaction between TNFα and TNFα receptors. SPD-304 may employ
a similar mechanism of inhibition.
Structure-Based Design of Tricyclic NF-κB Inducing Kinase (NIK) Inhibitors That Have High Selectivity over Phosphoinositide-3-kinase (PI3K)
Author: Castanedo GM, Blaquiere N, Beresini M, Bravo B, Brightbill H, Chen J, Cui HF, Eigenbrot C, Everett C, Feng J, Godemann R, Gogol E, Hymowitz S, Johnson A, Kayagaki N, Kohli PB, Knüppel K, Kraemer J, Krüger S, Loke P, McEwan P, Montalbetti C, Roberts DA, Smith M, Steinbacher S, Sujatha-Bhaskar S, Takahashi R, Wang X, Wu LC, Zhang Y, Staben ST.
Publication with: Genentech, Inc.
Citation: J Med Chem. 2017 Jan 26;60(2):627-640. doi: 10.1021/acs.jmedchem.6b01363. Epub 2017 Jan 12.
Abstract:
We report here structure-guided optimization of a novel series of NF-κB inducing
kinase (NIK) inhibitors. Starting from a modestly potent, low molecular weight
lead, activity was improved by designing a type 11/2 binding mode that accessed
a back pocket past the methionine-471 gatekeeper. Divergent binding modes in NIK
and PI3K were exploited to dampen PI3K inhibition while maintaining NIK
inhibition within these series. Potent compounds were discovered that
selectively inhibit the nuclear translocation of NF-κB2 (p52/REL-B) but not
canonical NF-κB1 (REL-A/p50).
Kinetic and structural insights into the binding of histone deacetylase 1 and 2 (HDAC1, 2) inhibitors
Author: Wagner FF, Weïwer M, Steinbacher S, Schomburg A, Reinemer P, Gale JP, Campbell AJ, Fisher SL, Zhao WN, Reis SA, Hennig KM, Thomas M, Müller P, Jefson MR, Fass DM, Haggarty SJ, Zhang YL, Holson EB.
Publication with: Broad Institute of MIT and Harvard
Citation: Bioorg Med Chem. 2016 Sep 15;24(18):4008-4015. doi: 10.1016/j.bmc.2016.06.040. Epub 2016 Jun 22.
Abstract:
The structure-activity and structure-kinetic relationships of a series of novel
and selective ortho-aminoanilide inhibitors of histone deacetylases (HDACs) 1
and 2 are described. Different kinetic and thermodynamic selectivity profiles
were obtained by varying the moiety occupying an 11Å channel leading to the
Zn(2+) catalytic pocket of HDACs 1 and 2, two paralogs with a high degree of
structural similarity. The design of these novel inhibitors was informed by two
ligand-bound crystal structures of truncated hHDAC2. BRD4884 and BRD7232 possess
kinetic selectivity for HDAC1 versus HDAC2. We demonstrate that the binding
kinetics of HDAC inhibitors can be tuned for individual isoforms in order to
modulate target residence time while retaining functional activity and increased
histone H4K12 and H3K9 acetylation in primary mouse neuronal cell culture
assays. These chromatin modifiers, with tuned binding kinetic profiles, can be
used to define the relation between target engagement requirements and the
pharmacodynamic response of HDACs in different disease applications.
Design and Development of a Series of Potent and Selective Type II Inhibitors of CDK8
Author: Bergeron P, Koehler MF, Blackwood EM, Bowman K, Clark K, Firestein R, Kiefer JR, Maskos K, McCleland ML, Orren L, Ramaswamy S, Salphati L, Schmidt S, Schneider EV, Wu J, Beresini M.
Publication with: Genentech, Inc.
Citation: ACS Med Chem Lett. 2016 Apr 5;7(6):595-600. doi: 10.1021/acsmedchemlett.6b00044. eCollection 2016 Jun 9.
Abstract:
Using Sorafenib as a starting point, a series of potent and selective inhibitors
of CDK8 was developed. When cocrystallized with CDK8 and cyclin C, these compounds exhibit a Type-II (DMG-out) binding mode.
Structural and biochemical characterization of the cell fate determining nucleotidyltransferase fold protein MAB21L1
Author: de Oliveira Mann CC, Kiefersauer R, Witte G, Hopfner KP.
Publication with: Ludwig-Maximilians Universität München
Citation: Sci Rep. 2016 Jun 8;6:27498. doi: 10.1038/srep27498.
Abstract:
The exceptionally conserved metazoan MAB21 proteins are implicated in cell fate
decisions and share considerable sequence homology with the cyclic GMP-AMP
synthase. cGAS is the major innate immune sensor for cytosolic DNA and produces
the second messenger 2'-5', 3'-5' cyclic GMP-AMP. Little is known about the
structure and biochemical function of other proteins of the cGAS-MAB21
subfamily, such as MAB21L1, MAB21L2 and MAB21L3. We have determined the crystal
structure of human full-length MAB21L1. Our analysis reveals high structural
conservation between MAB21L1 and cGAS but also uncovers important differences.
Although monomeric in solution, MAB21L1 forms a highly symmetric
double-pentameric oligomer in the crystal, raising the possibility that
oligomerization could be a feature of MAB21L1. In the crystal, MAB21L1 is in an
inactive conformation requiring a conformational change - similar to cGAS - to
develop any nucleotidyltransferase activity. Co-crystallization with NTP
identified a putative ligand binding site of MAB21 proteins that corresponds to
the DNA binding site of cGAS. Finally, we offer a structure-based explanation
for the effects of MAB21L2 mutations in patients with eye malformations. The
underlying residues participate in fold-stabilizing interaction networks and
mutations destabilize the protein. In summary, we provide a first structural
framework for MAB21 proteins.
Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site
Author: Chen H, Tucker J, Wang X, Gavine PR, Phillips C, Augustin MA, Schreiner P, Steinbacher S, Preston M, Ogg D.
Publication with: AstraZeneca R&D
Citation: Acta Crystallogr D Struct Biol. 2016 May;72(Pt 5):682-93. doi: 10.1107/S2059798316004502. Epub 2016 Apr 26.
Abstract:
MAP kinases act as an integration point for multiple biochemical signals and are
involved in a wide variety of cellular processes such as proliferation,
differentiation, regulation of transcription and development. As a member of the
MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling
pathways of various cell-surface receptors, including receptor tyrosine kinases
and G protein-coupled receptors. In the current study, five structures of the
ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported.
Interestingly, three of the compounds bind at a novel allosteric binding site in
ERK5, while the other two bind at the typical ATP-binding site. Binding of
inhibitors at the allosteric site is accompanied by displacement of the P-loop
into the ATP-binding site and is shown to be ATP-competitive in an enzymatic
assay of ERK5 kinase activity. Kinase selectivity data show that the most potent
allosteric inhibitor exhibits superior kinase selectivity compared with the two
inhibitors that bind at the canonical ATP-binding site. An analysis of these
structures and comparison with both a previously published ERK5-inhibitor
complex structure (PDB entry 4b99) and the structures of three other kinases
(CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.
Development of a Potent, Specific CDK8 Kinase Inhibitor Which Phenocopies CDK8/19 Knockout Cells
Author: Koehler MF, Bergeron P, Blackwood EM, Bowman K, Clark KR, Firestein R, Kiefer JR, Maskos K, McCleland ML, Orren L, Salphati L, Schmidt S, Schneider EV, Wu J, Beresini MH.
Publication with: Genentech, Inc.
Citation: ACS Med Chem Lett. 2016 Jan 6;7(3):223-8. doi: 10.1021/acsmedchemlett.5b00278. eCollection 2016 Mar 10.
Abstract:
Beginning with promiscuous COT inhibitors, which were found to inhibit CDK8, a
series of 6-aza-benzothiophene containing compounds were developed into potent,
selective CDK8 inhibitors. When cocrystallized with CDK8 and cyclin C, these
compounds exhibit an unusual binding mode, making a single hydrogen bond to the
hinge residue A100, a second to K252, and a key cation-π interaction with R356.
Structure-based drug design resulted in tool compounds 13 and 32, which are
highly potent, kinase selective, permeable compounds with a free fraction >2%
and no measurable efflux. Despite these attractive properties, these compounds
exhibit weak antiproliferative activity in the HCT-116 colon cancer cell line.
Further examination of the activity of 32 in this cell line revealed that the
compound reduced phosphorylation of the known CDK8 substrate STAT1 in a manner
identical to a CDK8 knockout clone, illustrating the complex effects of
inhibition of CDK8 kinase activity in proliferation in these cells.
Data for the crystal structure of APRIL-BAFF-BAFF heterotrimer
Author: Maskos K, Lammens A, Tan SL, Hess H, Palinsky W, Schneider P, Jiang X.
Publication with: EMD Serono Research & Development Institute
Citation: Data Brief. 2015 Dec 18;6:438-44. doi: 10.1016/j.dib.2015.12.024. eCollection 2016 Mar.
Abstract:
The TNF family ligands B cell activation factor (BAFF) and a
proliferation-inducing ligand (APRIL) modulate B cell function by forming
homotrimers and heterotrimers. To determine the structure of a heterotrimer of
BAFF and APRIL, these ligands were expressed as a single chain protein in HEK
293 cells, purified by affinity and size exclusion chromatographies, and
crystallized. Crystals belonging to the orthorhombic crystal system with a space
group of C2221 diffracted to 2.43 Å. Initial structural solution was obtained by
the molecular replacement method, and the structure was further refined to an R
factor of 0.179 and free R factor of 0.234. The atomic coordinates and structure
factors have been deposited into the Protein Data Bank (accession code 4ZCH).
The Rational Design of Selective Benzoxazepin Inhibitors of the α-Isoform of Phosphoinositide 3-Kinase Culminating in the Identification of (S)-2-((2-(1-Isopropyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)oxy)propanamide (GDC-0326)
Author: Heffron TP, Heald RA, Ndubaku C, Wei B, Augistin M, Do S, Edgar K, Eigenbrot C, Friedman L, Gancia E, Jackson PS, Jones G, Kolesnikov A, Lee LB, Lesnick JD, Lewis C, McLean N, Mörtl M, Nonomiya J, Pang J, Price S, Prior WW, Salphati L, Sideris S, Staben ST, Steinbacher S, Tsui V, Wallin J, Sampath D, Olivero AG.
Publication with: Genentech, Inc.
Citation: J Med Chem. 2016 Feb 11;59(3):985-1002. doi: 10.1021/acs.jmedchem.5b01483. Epub 2016 Jan 20.
Abstract:
Inhibitors of the class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have
received substantial attention for their potential use in cancer therapy.
Despite the particular attraction of targeting PI3Kα, achieving selectivity for
the inhibition of this isoform has proved challenging. Herein we report the
discovery of inhibitors of PI3Kα that have selectivity over the other class I
isoforms and all other kinases tested. In GDC-0032 (3, taselisib), we previously
minimized inhibition of PI3Kβ relative to the other class I insoforms.
Subsequently, we extended our efforts to identify PI3Kα-specific inhibitors
using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors
with selectivity for PI3Kα through interactions with a nonconserved residue.
Several molecules selective for PI3Kα relative to the other class I isoforms, as
well as other kinases, were identified. Optimization of properties related to
drug metabolism then culminated in the identification of the clinical candidate
GDC-0326 (4).
On-rate based optimization of structure-kinetic relationship--surfing the kinetic map
Author: Schoop A, Dey F.
Publication with: Roche Innovation Center
Citation: Drug Discov Today Technol. 2015 Oct;17:9-15. doi: 10.1016/j.ddtec.2015.08.003. Epub 2015 Sep 18.
Abstract:
In the lead discovery process residence time has become an important parameter
for the identification and characterization of the most efficacious compounds in
vivo. To enable the success of compound optimization by medicinal chemistry
toward a desired residence time the understanding of structure-kinetic
relationship (SKR) is essential. This article reviews various approaches to
monitor SKR and suggests using the on-rate as the key monitoring parameter. The
literature is reviewed and examples of compound series with low variability as
well as with significant changes in on-rates are highlighted. Furthermore,
findings of kinetic on-rate changes are presented and potential underlying
rationales are discussed.
Identification of azabenzimidazoles as potent JAK1 selective inhibitors
Author: Vasbinder MM, Alimzhanov M, Augustin M, Bebernitz G, Bell K, Chuaqui C, Deegan T, Ferguson AD, Goodwin K, Huszar D, Kawatkar A, Kawatkar S, Read J, Shi J, Steinbacher S, Steuber H, Su Q, Toader D, Wang H, Woessner R, Wu A, Ye M, Zinda M.
Publication with: AstraZeneca R&D
Citation: Bioorg Med Chem Lett. 2016 Jan 1;26(1):60-7. doi: 10.1016/j.bmcl.2015.11.031. Epub 2015 Nov 12.
Abstract:
We have identified a class of azabenzimidazoles as potent and selective JAK1
inhibitors. Investigations into the SAR are presented along with the structural
features required to achieve selectivity for JAK1 versus other JAK family
members. An example from the series demonstrated highly selective inhibition of
JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it
to serve as a JAK1 selective tool compound to further probe the biology of JAK1
selective inhibitors.
Design, Synthesis, and Biological Evaluation of Quercetagetin Analogues as JNK1 Inhibitors
Author: Hierold J, Baek S, Rieger R, Lim TG, Zakpur S, Arciniega M, Lee KW, Huber R, Tietze LF.
Publication with: Max-Planck-Institute for Biochemistry
Citation: Chemistry. 2015 Nov 16;21(47):16887-94. doi: 10.1002/chem.201502475. Epub 2015 Oct 7.
Abstract:
The recent discovery of c-Jun NH2-terminal kinase JNK1 suppression by natural quercetagetin (1) is a promising lead for the development of novel anticancer agents. Using both X-ray structure and docking analyses we predicted that 5'-hydroxy- (2) and 5'-hydroxymethyl-quercetagetin (3) would inhibit JNK1 more actively than the parent compound 1. Notably, our drug design was based on the active enzyme-ligand complex as opposed to the enzyme's relatively open apo
structure. In this paper we test our theoretical predictions, aided by
docking-model experiments, and report the first synthesis and biological
evaluation of quercetagetin analogues 2 and 3. As calculated, both compounds strongly suppress JNK1 activity. The IC50 values were determined to be 3.4 μM
and 12.2 μM, respectively, which shows that 2 surpasses the potency of the parent compound 1 (IC50 =4.6 μM). Compound 2 was also shown to suppress matrix metalloproteinase-1 expression with high specificity after UV irradiation.
Mediator kinase inhibition further activates super-enhancer-associated genes in AML
Author: Pelish HE, Liau BB, Nitulescu II, Tangpeerachaikul A, Poss ZC, Da Silva DH, Caruso BT, Arefolov A, Fadeyi O, Christie AL, Du K, Banka D, Schneider EV, Jestel A, Zou G, Si C, Ebmeier CC, Bronson RT, Krivtsov AV, Myers AG, Kohl NE, Kung AL, Armstrong SA, Lemieux ME, Taatjes DJ, Shair MD.
Publication with: Harvard University
Citation: Nature. 2015 Oct 8;526(7572):273-276. doi: 10.1038/nature14904. Epub 2015 Sep 28.
Abstract:
Super-enhancers (SEs), which are composed of large clusters of enhancers densely
loaded with the Mediator complex, transcription factors and chromatin
regulators, drive high expression of genes implicated in cell identity and
disease, such as lineage-controlling transcription factors and oncogenes. BRD4
and CDK7 are positive regulators of SE-mediated transcription. By contrast,
negative regulators of SE-associated genes have not been well described. Here we
show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and
CDK19 restrain increased activation of key SE-associated genes in acute myeloid
leukaemia (AML) cells. We report that the natural product cortistatin A (CA)
selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and
in vivo, and disproportionately induces upregulation of SE-associated genes in
CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells,
CA upregulated SE-associated genes with tumour suppressor and
lineage-controlling functions, including the transcription factors CEBPA, IRF8,
IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these
SE-associated genes, yet also has anti-leukaemic activity. Individually
increasing or decreasing the expression of these transcription factors
suppressed AML cell growth, providing evidence that leukaemia cells are
sensitive to the dosage of SE-associated genes. Our results demonstrate that
Mediator kinases can negatively regulate SE-associated gene expression in
specific cell types, and can be pharmacologically targeted as a therapeutic
approach to AML.
Production of aggregation prone human interferon gamma and its mutant in highly soluble and biologically active form by SUMO fusion technology
Author: Tileva M, Krachmarova E, Ivanov I, Maskos K, Nacheva G.
Publication with: Bulgarian Academy of Sciences, Bulgaria
Citation: Protein Expr Purif. 2016 Jan;117:26-34. doi: 10.1016/j.pep.2015.09.022. Epub 2015 Sep 25.
Abstract:
The Escherichia coli expression system is a preferable choice for production of
recombinant proteins. A disadvantage of this system is the target protein
aggregation in "inclusion bodies" (IBs) that further requires solubilisation and
refolding, which is crucial for the properties and the yield of the final
product. In order to prevent aggregation, SUMO fusion tag technology has been
successfully applied for expression of eukaryotic proteins, including human
interferon gamma (hIFNγ) that was reported, however, with no satisfactory
biological activity. We modified this methodology for expression and
purification of both the wild type hIFNγ and an extremely prone to aggregation
mutant hIFNγ-K88Q, whose recovery from IBs showed to be ineffective upon
numerous conditions. By expression of the N-terminal His-SUMO fusion proteins in
the E. coli strain BL21(DE3)pG-KJE8, co-expressing two chaperone systems, at 24
°C a significant increase in solubility of both target proteins (1.5-fold for
hIFNγ and 8-fold for K88Q) was achieved. Two-step chromatography (affinity and
ion-exchange) with on-dialysis His-SUMO-tag cleavage was applied for protein
purification that yielded 6.0-7.0mg/g wet biomass for both proteins with >95%
purity and native N-termini. The optimised protocol led to increased yields from
5.5 times for hIFNγ up to 100 times for K88Q in comparison to their isolation
from IBs. Purified hIFNγ showed preserved thermal stability and
antiproliferative activity corresponding to that of the native reference sample
(3 × 10(7)IU/mg). The developed methodology represents an optimised procedure
that can be successfully applied for large scale expression and purification of
aggregation-prone proteins in soluble native form.
Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti-human GM-CSF antibody
Author: Eylenstein R, Weinfurtner D, Härtle S, Strohner R, Böttcher J, Augustin M, Ostendorp R, Steidl S.
Publication with: Boehringer Ingelheim RCV GmbH & Co KG
Citation: MAbs. 2016;8(1):176-86. doi: 10.1080/19420862.2015.1099774. Epub 2015 Sep 25.
Abstract:
X-ray structure analysis of 4 antibody Fab fragments, each in complex with human
granulocyte macrophage colony stimulating factor (GM-CSF), was performed to
investigate the changes at the protein-protein binding interface during the
course of in vitro affinity maturation by phage display selection. The parental
antibody MOR03929 was compared to its derivatives MOR04252 (CDR-H2 optimized),
MOR04302 (CDR-L3 optimized) and MOR04357 (CDR-H2 and CDR-L3 optimized). All
antibodies bind to a conformational epitope that can be divided into 3
sub-epitopes. Specifically, MOR04357 binds to a region close to the GM-CSF
N-terminus (residues 11-24), a short second sub-epitope (residues 83-89) and a
third at the C-terminus (residues 112-123). Modifications introduced during
affinity maturation in CDR-H2 and CDR-L3 led to the establishment of additional
hydrogen bonds and van der Waals contacts, respectively, providing a rationale
for the observed improvement in binding affinity and neutralization potency.
Once GM-CSF is complexed to the antibodies, modeling predicts a sterical clash
with GM-CSF binding to GM-CSF receptor α and β chain. This predicted mutually
exclusive binding was confirmed by a GM-CSF receptor α chain ligand binding
inhibition assay. Finally, high throughput sequencing of clones obtained after
affinity maturation phage display pannings revealed highly selected consensus
sequences for CDR-H2 as well for CDR-L3, which are in accordance with the
sequence of the highest affinity antibody MOR04357. The resolved crystal
structures highlight the criticality of these strongly selected residues for
high affinity interaction with GM-CSF.
Flt3 is a target of coumestrol in protecting against UVB-induced skin photoaging
Author: Park G, Baek S, Kim JE, Lim TG, Lee CC, Yang H, Kang YG, Park JS, Augustin M, Mrosek M, Lee CY, Dong Z, Huber R, Lee KW.
Publication with: Seoul National University, Korea
Citation: Biochem Pharmacol. 2015 Dec 1;98(3):473-83. doi: 10.1016/j.bcp.2015.08.104. Epub 2015 Sep 2.
Abstract:
While skin aging is a naturally occurring process by senescence, exposure to ultraviolet (UV) radiation accelerates wrinkle formation and sagging of skin. UV induces skin aging by degrading collagen via activating matrix metalloproteinases (MMPs). In this study, we show that coumestrol, a metabolite of the soybean isoflavone daidzein, has a preventive effect on skin photoaging in three-dimensional human skin equivalent model. Coumestrol inhibited UVB-induced MMP-1 expression and activity. Whole human kinase profiling assay identified FLT3 kinase as a novel target protein of coumestrol in UVB-induced
signaling pathway in skin. Coumestrol suppresses FLT3 kinase activity, and
subsequently, Ras/MEK/ERK and Akt/p70 ribosomal S6 kinase pathway. This
suppresses AP-1 activity and in turn, diminishes MMP-1 gene transcription. Using X-ray crystallography, the binding of coumestrol to FLT3 was defined and implied ATP-competitive inhibition. Residues Lys644 and Phe830 showed local changes to accommodate coumestrol in the ATP-binding pocket. 4-APIA, a pharmacological inhibitor of FLT3, inhibited MMP-1 expression and induced signal transduction
changes similar to coumestrol. Taken together, coumestrol inhibits UVB-induced MMP-1 expression by suppressing FLT3 kinase activity. These findings suggest that coumestrol is a novel dietary compound with potential application in
preventing and improving UVB-associated skin aging.
One Question, Multiple Answers: Biochemical and Biophysical Screening Methods Retrieve Deviating Fragment Hit Lists
Author: Schiebel J, Radeva N, Köster H, Metz A, Krotzky T, Kuhnert M, Diederich WE, Heine A, Neumann L, Atmanene C, Roecklin D, Vivat-Hannah V, Renaud JP, Meinecke R, Schlinck N, Sitte A, Popp F, Zeeb M, Klebe G.
Publication with: NovAliX
Citation: ChemMedChem. 2015 Sep;10(9):1511-21. doi: 10.1002/cmdc.201500267. Epub 2015 Aug 10.
Abstract:
Fragment-based lead discovery is gaining momentum in drug development.
Typically, a hierarchical cascade of several screening techniques is consulted
to identify fragment hits which are then analyzed by crystallography. Because
crystal structures with bound fragments are essential for the subsequent
hit-to-lead-to-drug optimization, the screening process should distinguish
reliably between binders and non-binders. We therefore investigated whether
different screening methods would reveal similar collections of putative
binders. First we used a biochemical assay to identify fragments that bind to
endothiapepsin, a surrogate for disease-relevant aspartic proteases. In a
comprehensive screening approach, we then evaluated our 361-entry library by
using a reporter-displacement assay, saturation-transfer difference NMR, native
mass spectrometry, thermophoresis, and a thermal shift assay. While the combined
results of these screening methods retrieve 10 of the 11 crystal structures
originally predicted by the biochemical assay, the mutual overlap of individual
hit lists is surprisingly low, highlighting that each technique operates on
different biophysical principles and conditions.
Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties
Author: Schuepbach-Mallepell S, Das D, Willen L, Vigolo M, Tardivel A, Lebon L, Kowalczyk-Quintas C, Nys J, Smulski C, Zheng TS, Maskos K, Lammens A, Jiang X, Hess H, Tan SL, Schneider P.
Publication with: Merck KGaA,
Citation: J Biol Chem. 2015 Jun 26;290(26):16330-42. doi: 10.1074/jbc.M115.661405. Epub 2015 May 7.
Abstract:
The closely related TNF family ligands B cell activation factor (BAFF) and a
proliferation-inducing ligand (APRIL) serve in the generation and maintenance of
mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and
activate several receptors that they partially share. However, heteromers of
BAFF and APRIL that occur in patients with autoimmune diseases are incompletely
characterized. The N and C termini of adjacent BAFF or APRIL monomers are
spatially close and can be linked to create single-chain homo- or hetero-ligands
of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF
and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA),
transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor
(BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and
BCMA and weakly to BAFFR in accordance with the analysis of the receptor
interaction sites in the crystallographic structure of ABB. Receptor binding
correlated with activity in reporter cell line assays specific for BAFFR, TACI,
or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but
not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in
BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different
stoichiometries have distinct receptor-binding properties and activities. Based
on the observation that heteromers are less active than BAFF, we speculate that
their physiological role might be to down-regulate BAFF activity.
Distinct binding mode of multikinase inhibitor lenvatinib revealed by biochemical characterization
Author: Okamoto K, Ikemori-Kawada M, Jestel A, von König K, Funahashi Y, Matsushima T, Tsuruoka A, Inoue A, Matsui J.
Publication with: Eisai, Inc.
Citation: ACS Med Chem Lett. 2014 Nov 17;6(1):89-94. doi: 10.1021/ml500394m. eCollection 2015 Jan 8.
Abstract:
Lenvatinib is an oral multikinase inhibitor that selectively inhibits vascular
endothelial growth factor (VEGF) receptors 1 to 3 and other proangiogenic and
oncogenic pathway-related receptor tyrosine kinases. To elucidate the origin of
the potency of lenvatinib in VEGF receptor 2 (VEGFR2) inhibition, we conducted a
kinetic interaction analysis of lenvatinib with VEGFR2 and X-ray analysis of the
crystal structure of VEGFR2-lenvatinib complexes. Kinetic analysis revealed that
lenvatinib had a rapid association rate constant and a relatively slow
dissociation rate constant in complex with VEGFR2. Co-crystal structure analysis
demonstrated that lenvatinib binds at its ATP mimetic quinoline moiety to the
ATP binding site and to the neighboring region via a cyclopropane ring, adopting
an Asp-Phe-Gly (DFG)-"in" conformation. These results suggest that lenvatinib is
very distinct in its binding mode of interaction compared to the several
approved VEGFR2 kinase inhibitors.
Structural analysis of the human fibroblast growth factor receptor 4 kinase
Author: Lesca E, Lammens A, Huber R, Augustin M.
Publication with: Max Planck Institute of Biochemistry
Citation: J Mol Biol. 2014 Nov 11;426(22):3744-3756. doi: 10.1016/j.jmb.2014.09.004. Epub 2014 Sep 16.
Abstract:
The family of fibroblast growth factor receptors (FGFRs) plays an important and
well-characterized role in a variety of pathological disorders. FGFR4 is
involved in myogenesis and muscle regeneration. Mutations affecting the kinase
domain of FGFR4 may cause cancer, for example, breast cancer or
rhabdomyosarcoma. Whereas FGFR1-FGFR3 have been structurally characterized, the
structure of the FGFR4 kinase domain has not yet been reported. In this study,
we present four structures of the kinase domain of FGFR4, in its apo-form and in
complex with different types of small-molecule inhibitors. The two apo-FGFR4
kinase domain structures show an activation segment similar in conformation to
an autoinhibitory segment observed in the hepatocyte growth factor receptor
kinase but different from the known structures of other FGFR kinases. The
structures of FGFR4 in complex with the type I inhibitor Dovitinib and the type
II inhibitor Ponatinib reveal the molecular interactions with different types of
kinase inhibitors and may assist in the design and development of FGFR4
inhibitors.
Structure-based optimization of non-peptidic Cathepsin D inhibitors
Author: Grädler U, Czodrowski P, Tsaklakidis C, Klein M, Werkmann D, Lindemann S, Maskos K, Leuthner B.
Publication with: Merck KGaA
Citation: Bioorg Med Chem Lett. 2014 Sep 1;24(17):4141-50. doi: 10.1016/j.bmcl.2014.07.054. Epub 2014 Jul 25.
Abstract:
We discovered a novel series of non-peptidic acylguanidine inhibitors of
Cathepsin D as target for osteoarthritis. The initial HTS-hits were optimized by
structure-based design using CatD X-ray structures resulting in single digit
nanomolar potency in the biochemical CatD assay. However, the most potent
analogues showed only micromolar activities in an ex vivo glycosaminoglycan
(GAG) release assay in bovine cartilage together with low cellular permeability
and suboptimal microsomal stability. This new scaffold can serve as a starting
point for further optimization towards in vivo efficacy.
IR laser-induced protein crystal transformation
Author: Kiefersauer R, Grandl B, Krapp S, Huber R.
Publication with: Max-Planck-Institut für Biochemie
Citation: Acta Crystallogr D Biol Crystallogr. 2014 May;70(Pt 5):1224-32. doi: 10.1107/S1399004714002223. Epub 2014 Apr 26.
Abstract:
A method and the design of instrumentation, and its preliminary practical
realisation, including test experiments, with the object of inducing phase
changes of biomolecular crystals by controlled dehydration through heating with
infrared (IR) light are described. The aim is to generate and select crystalline
phases through transformation in the solid state which have improved order
(higher resolution in X-ray diffraction experiments) and reduced mosaic spread
(more uniformly aligned mosaic blocks) for diffraction data collection and
analysis. The crystal is heated by pulsed and/or constant IR laser irradiation.
Loss of crystal water following heating and its reabsorption through
equilibration with the environment is measured optically by a video system.
Heating proved superior to traditional controlled dehydration by humidity change
for the test cases CODH (carbon monoxide dehydrogenase) and CLK2 (a protein
kinase). Heating with IR light is experimentally simple and offers an
exploration of a much broader parameter space than the traditional method, as it
allows the option of varying the rate of phase changes through modification of
the IR pulse strength, width and repeat frequency. It impacts the crystal
instantaneously, isotropically and homogeneously, and is therefore expected to
cause less mechanical stress.
Crystal structure of human TWEAK in complex with the Fab fragment of a neutralizing antibody reveals insights into receptor binding
Author: Lammens A, Baehner M, Kohnert U, Niewoehner J, von Proff L, Schraeml M, Lammens K, Hopfner KP.
Publication with: Center for Integrated Protein Science-CIPSM, Germany
Citation: PLoS One. 2013 May 8;8(5):e62697. doi: 10.1371/journal.pone.0062697. Print 2013.
Abstract:
The tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a
multifunctional cytokine playing a key role in tissue regeneration and
remodeling. Dysregulation of TWEAK signaling is involved in various pathological
processes like autoimmune diseases and cancer. The unique interaction with its
cognate receptor Fn14 makes both ligand and receptor promising targets for novel
therapeutics. To gain insights into this important signaling pathway, we
determined the structure of soluble human TWEAK in complex with the Fab fragment
of an antibody selected for inhibition of receptor binding. In the crystallized
complex TWEAK is bound by three Fab fragments of the neutralizing antibody.
Homology modeling shows that Fab binding overlaps with the putative Fn14 binding
site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD) to that site
generates a highly complementary interface with perfectly opposing charged and
hydrophobic residues. Taken together the presented structure provides new
insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help
to optimize the therapeutic strategy for treatment of related cancer types and
autoimmune diseases.
An old NSAID revisited: crystal structure of aldose reductase in complex with sulindac at 1.0 Å supports a novel mechanism for its anticancer and antiproliferative effects
Author: Steuber H.
Publication with: Proteros Biostructures GmbH
Citation: ChemMedChem. 2011 Dec 9;6(12):2155-7. doi: 10.1002/cmdc.201100374. Epub 2011 Oct 13.
Abstract:
Key factors for successful generation of protein-fragment structures requirement on protein, crystals, and technology
Author: Böttcher J, Jestel A, Kiefersauer R, Krapp S, Nagel S, Steinbacher S, Steuber H.
Publication with: Proteros Biostructures GmbH
Citation: Methods Enzymol. 2011;493:61-89. doi: 10.1016/B978-0-12-381274-2.00003-0.
Abstract:
In the past two decades, fragment-based approaches have evolved as a predominant
strategy in lead discovery. The availability of structural information on the
interaction geometries of binding fragments is key to successful
structure-guided fragment-to-lead evolution. In this chapter, we illustrate
methodological advances for protein-fragment crystal structure generation in
order to offer general lessons on the importance of fragment properties and the
most appropriate crystallographic setup to evaluate them. We analyze elaborate
protocols, methods, and clues applied to challenging complex formation projects.
The results should assist medicinal chemists to select the most promising
targets and strategies for fragment-based crystallography as well as provide a
tutorial to structural biologists who attempt to determine protein-fragment
structures.
HTS reporter displacement assay for fragment screening and fragment evolution toward leads with optimized binding kinetics, binding selectivity, and thermodynamic signature
Author: Neumann L, von König K, Ullmann D.
Publication with: Proteros Biostructures GmbH
Citation: Methods Enzymol. 2011;493:299-320. doi: 10.1016/B978-0-12-381274-2.00012-1.
Abstract:
Parameters such as residence time, kinetic selectivity, and thermodynamic
signature are more and more under debate as optimization objectives within
fragment-based lead discovery. However, broad implementation of these parameters
is hampered by the lack of technologies that give rapid access to binding
kinetics and thermodynamic information for large amounts of compound-target
interactions. Here, the authors describe a technology--the reporter displacement
assay--that is capable of opening this bottleneck and of supporting data-driven
design of lead compounds with tailor-made residence time, kinetic selectivity,
and thermodynamic signature.
Fragment-based lead generation: identification of seed fragments by a highly efficient fragment screening technology
Author: Neumann L, Ritscher A, Müller G, Hafenbradl D.
Publication with: Proteros Biostructures GmbH
Citation: J Comput Aided Mol Des. 2009 Aug;23(8):501-11. doi: 10.1007/s10822-009-9288-x. Epub 2009 Jun 17.
Abstract:
For the detection of the precise and unambiguous binding of fragments to a
specific binding site on the target protein, we have developed a novel reporter
displacement binding assay technology. The application of this technology for
the fragment screening as well as the fragment evolution process with a specific
modelling based design strategy is demonstrated for inhibitors of the protein
kinase p38alpha. In a fragment screening approach seed fragments were identified
which were then used to build compounds from the deep-pocket towards the hinge
binding area of the protein kinase p38alpha based on a modelling approach.
BIRB796 was used as a blueprint for the alignment of the fragments. The fragment
evolution of these deep-pocket binding fragments towards the fully optimized
inhibitor BIRB796 included the modulation of the residence time as well as the
affinity. The goal of our study was to evaluate the robustness and efficiency of
our novel fragment screening technology at high fragment concentrations, compare
the screening data with biochemical activity data and to demonstrate the
evolution of the hit fragments with fast kinetics, into slow kinetic inhibitors
in an in silico approach.
Catalysis at a dinuclear [CuSMo(==O)OH] cluster in a CO dehydrogenase resolved at 1.1-A resolution
Author: Dobbek H, Gremer L, Kiefersauer R, Huber R, Meyer O.
Publication with: Max Planck Institute of Biochemistry
Citation: Proc Natl Acad Sci U S A. 2002 Dec 10;99(25):15971-6. doi: 10.1073/pnas.212640899.
Abstract:
The CO dehydrogenase of the eubacterium Oligotropha carboxidovorans is a 277-kDa
Mo- and Cu-containing iron-sulfur flavoprotein. Here, the enzyme's active site
in the oxidized or reduced state, after inactivation with potassium cyanide or
with n-butylisocyanide bound to the active site, has been reinvestigated by
multiple wavelength anomalous dispersion measurements at atomic resolution,
electron spin resonance spectroscopy, and chemical analyses. We present evidence
for a dinuclear heterometal [CuSMoO)OH] cluster in the active site of the
oxidized or reduced enzyme, which is prone to cyanolysis. The cluster is
coordinated through interactions of the Mo with the dithiolate pyran ring of
molybdopterin cytosine dinucleotide and of the Cu with the Sgamma of Cys-388,
which is part of the active-site loop VAYRC(388)SFR. The previously reported
active-site structure [Dobbek, H., Gremer, L., Meyer, O. & Huber, R. (1999)
Proc. Natl. Acad. Sci. USA 96, 8884-8889] of an Mo with three oxygen ligands and
an SeH-group bound to the Sgamma atom of Cys-388 could not be confirmed. The
structure of CO dehydrogenase with the inhibitor n-butylisocyanide bound has led
to a model for the catalytic mechanism of CO oxidation which involves a
thiocarbonate-like intermediate state. The dinuclear [CuSMo(O)OH] cluster of CO
dehydrogenase establishes a previously uncharacterized class of dinuclear
molybdoenzymes containing the pterin cofactor.
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