scholarly journals Photochemical probe identification of the small-molecule binding site in a mammalian membrane-bound O-acyltransferase

2020 ◽  
Author(s):  
Thomas Lanyon-Hogg ◽  
Markus Ritzefeld ◽  
Leran Zhang ◽  
Balazs Pogranyi ◽  
Milon Mondal ◽  
...  
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Drug Targets ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Inhibitory Potency ◽  
Interaction Sites ◽  
Small Molecule Inhibition ◽  
Membrane Bound ◽  
Hedgehog Acyltransferase

AbstractThe mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports development of a photochemical probe to interrogate the small-molecule binding site in the human MBOAT Hedgehog acyltransferase (HHAT) based on HHAT inhibitor RUSKI-201. Structure-activity relationship investigation identified the improved enantiomeric inhibitor IMP-1575, which is the most potent HHAT inhibitor reported to-date, and guided rational design of a photocrosslinking probe that maintained HHAT-inhibitory potency. Photocrosslinking and proteomic sequencing of HHAT delivered identification of the first small-molecule binding site in a mammalian MBOAT. Topology and homology data suggested a potential mechanism for HHAT inhibition which was confirmed via kinetic analysis. Our results provide an optimal HHAT inhibitor IMP-1575 (Ki = 38 nM) and a strategy for mapping of interaction sites in MBOATs.

scholarly journals Photochemical probe identification of a small‐molecule inhibitor binding site in Hedgehog acyltransferase (HHAT)

2021 ◽  
Author(s):  
Thomas Lanyon-Hogg ◽  
Markus Ritzefeld ◽  
Leran Zhang ◽  
Balazs Pogranyi ◽  
Milon Mondal ◽  
...  
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Small Molecule Inhibitor ◽  
Inhibitor Binding ◽  
Molecule Inhibitor ◽  
Hedgehog Acyltransferase

scholarly journals Mapping major SARS-CoV-2 drug targets and assessment of druggability using computational fragment screening: Identification of an allosteric small-molecule binding site on the Nsp13 helicase

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246181
Author(s):  
Matthew R. Freidel ◽  
Roger S. Armen
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Small Molecule ◽  
Binding Sites ◽  
Drug Targets ◽  
Human Life ◽  
Free Energy Calculations ◽  
Allosteric Site ◽  
Preclinical Development ◽  
Fragment Screening

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2’-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.

scholarly journals A small molecule inhibitor of HER3: a proof-of-concept study

2020 ◽  
Vol 477 (17) ◽  
pp. 3329-3347 ◽  
Author(s):  
Audrey Colomba ◽  
Martina Fitzek ◽  
Roger George ◽  
Gregory Weitsman ◽  
Selene Roberts ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Small Molecule ◽  
Drug Targets ◽  
Molecular Mechanisms ◽  
Growth Factor Receptor ◽  
Screening Strategy ◽  
Proof Of Concept ◽  
Allosteric Inhibitors ◽  
Concept Study ◽  
Molecule Inhibitor

Despite being catalytically defective, pseudokinases are typically essential players of cellular signalling, acting as allosteric regulators of their active counterparts. Deregulation of a growing number of pseudokinases has been linked to human diseases, making pseudokinases therapeutic targets of interest. Pseudokinases can be dynamic, adopting specific conformations critical for their allosteric function. Interfering with their allosteric role, with small molecules that would lock pseudokinases in a conformation preventing their productive partner interactions, is an attractive therapeutic strategy to explore. As a well-known allosteric activator of epidermal growth factor receptor family members, and playing a major part in cancer progression, the pseudokinase HER3 is a relevant context in which to address the potential of pseudokinases as drug targets for the development of allosteric inhibitors. In this proof-of-concept study, we developed a multiplex, medium-throughput thermal shift assay screening strategy to assess over 100 000 compounds and identify selective small molecule inhibitors that would trap HER3 in a conformation which is unfavourable for the formation of an active HER2–HER3 heterodimer. As a proof-of-concept compound, AC3573 bound with some specificity to HER3 and abrogated HER2–HER3 complex formation and downstream signalling in cells. Our study highlights the opportunity to identify new molecular mechanisms of action interfering with the biological function of pseudokinases.

scholarly journals Colchicine-Binding Site Inhibitors from Chemistry to Clinic: A Review

2020 ◽  
Vol 13 (1) ◽  
pp. 8 ◽  
Author(s):  
Eavan C. McLoughlin ◽  
Niamh M. O’Boyle
Keyword(s):  
Binding Site ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Rational Design ◽  
Clinical Status ◽  
Cancer Therapies ◽  
Colchicine Binding Site ◽  
Anti Cancer ◽  
Recent Developments ◽  
Important Drug

It is over 50 years since the discovery of microtubules, and they have become one of the most important drug targets for anti-cancer therapies. Microtubules are predominantly composed of the protein tubulin, which contains a number of different binding sites for small-molecule drugs. There is continued interest in drug development for compounds targeting the colchicine-binding site of tubulin, termed colchicine-binding site inhibitors (CBSIs). This review highlights CBSIs discovered through diverse sources: from natural compounds, rational design, serendipitously and via high-throughput screening. We provide an update on CBSIs reported in the past three years and discuss the clinical status of CBSIs. It is likely that efforts will continue to develop CBSIs for a diverse set of cancers, and this review provides a timely update on recent developments.

scholarly journals How does a small molecule bind at a cryptic binding site?

2021 ◽  
Author(s):  
Yibing Shan ◽  
Venkatesh P. Mysore ◽  
Abba E. Leffler ◽  
Eric T. Kim ◽  
Shiori Sagawa ◽  
...  
Keyword(s):  
Small Molecules ◽  
Binding Site ◽  
Small Molecule ◽  
Protein Interactions ◽  
Binding Sites ◽  
Rational Design ◽  
Structural Information ◽  
Interleukin 2 ◽  
Md Simulations ◽  
Drug Binding

Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are "cryptic": When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)—which performs its function by forming a PPI with its receptor—without incorporating any prior structural information about the ligands' binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein–small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2–small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites.

scholarly journals Small-molecule sensitization of RecBCD helicase–nuclease to a Chi hotspot-activated state

2020 ◽  
Vol 48 (14) ◽  
pp. 7973-7980
Author(s):  
Ahmet C Karabulut ◽  
Ryan T Cirz ◽  
Andrew F Taylor ◽  
Gerald R Smith
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Strand Exchange ◽  
Atp Binding ◽  
Dna Unwinding ◽  
Wild Type ◽  
Atp Binding Site ◽  
Activated State ◽  
Complex Enzymes

Abstract Coordinating multiple activities of complex enzymes is critical for life, including transcribing, replicating and repairing DNA. Bacterial RecBCD helicase–nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5′-ended strand and its slower RecB helicase on the 3′-ended strand. At Chi hotspots (5′ GCTGGTGG 3′), RecB’s nuclease cuts the 3′-ended strand and loads RecA strand-exchange protein onto it. We report that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate's length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB relative to RecD and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. ATP and NSAC1003 are competitive; computation docks NSAC1003 into RecB’s ATP-binding site, suggesting NSAC1003 acts directly on RecB. NSAC1003 will help elucidate molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes with activities coordinated at chromosomal sites.

scholarly journals Small-molecule sensitization of RecBCD helicase-nuclease to a Chi hotspot-activated state

2020 ◽  
Author(s):  
Ahmet C. Karabulut ◽  
Ryan T. Cirz ◽  
Gerald R. Smith
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Molecular Mechanisms ◽  
Strand Exchange ◽  
Atp Binding ◽  
Dna Unwinding ◽  
Wild Type ◽  
Atp Binding Site ◽  
Activated State ◽  
Complex Enzymes

ABSTRACTCoordination of multiple activities of complex enzymes is critical for life, including transcribing, replicating, and repairing DNA. Bacterial RecBCD helicase-nuclease must coordinate DNA unwinding and cutting to repair broken DNA. Starting at a DNA end, RecBCD unwinds DNA with its fast RecD helicase on the 5’-ended strand and its slower RecB helicase on the 3’-ended strand. At Chi hotspots (5’GCTGGTGG3’), RecB’s nuclease cuts the 3’-ended strand and loads RecA strand-exchange protein onto it. We report here that a small molecule NSAC1003, a sulfanyltriazolobenzimidazole, mimics Chi sites by sensitizing RecBCD to cut DNA at a Chi-independent position a certain percent of the DNA substrate’s length. This percent decreases with increasing NSAC1003 concentration. Our data indicate that NSAC1003 slows RecB and sensitizes it to cut DNA when the leading helicase RecD stops at the DNA distal end. Two previously described RecBCD mutants altered in the RecB ATP-binding site also have this property, but uninhibited wild-type RecBCD lacks it. Computation docks NSAC1003 into the ATP-binding site, suggesting that NSAC1003 acts directly on RecB. NSAC1003 will help elucidate the molecular mechanisms of RecBCD-Chi regulation and DNA repair. Similar studies could help elucidate other DNA enzymes whose activities are coordinated at chromosomal sites.

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