Receptor–Ligand Interaction-Based Virtual Screening for Novel Eg5/Kinesin Spindle Protein Inhibitors

Eg5/KSP is a promising mitotic spindle target for drug discovery in cancer chemotherapy and the development of agents against fungal diseases. A range of Eg5 targeting compounds identified by in vitro or cell-based screening is currently in development. We employed structure-based virtual screening of a database of 700 000 compounds to identify three novel Eg5 inhibitors bearing quinazoline (<b>24</b>) or thioxoimidazolidine (<b>30</b> and <b>37</b>) scaffolds. The new compounds inhibit Eg5 ATPase activity, show growth inhibition in proliferation assays, and induce monoastral spindles in cells, the characteristic phenotype for Eg5 inhibiting agents. This is the first successful reported procedure for the identification of Eg5 inhibitors via receptor–ligand interaction-based virtual screening

Mitotic Kinesin Eg5 Overcomes Inhibition to the Phase I/II Clinical Candidate SB743921 by an Allosteric Resistance Mechanism

Development of drug resistance during cancer chemotherapy is one of the major causes of chemotherapeutic failure for the majority of clinical agents. The aim of this study was to investigate the underlying molecular mechanism of resistance developed by the mitotic kinesin Eg5 against the potent second-generation ispinesib analogue SB743921 <b>(1)</b>, a phase I/II clinical candidate. Biochemical and biophysical data demonstrate that point mutations in the inhibitor-binding pocket decrease the efficacy of <b>1</b> by several 1000-fold. Surprisingly, the structures of wild-type and mutant Eg5 in complex with <b>1</b> display no apparent structural changes in the binding configuration of the drug candidate. Furthermore, ITC and modeling approaches reveal that resistance to <b>1</b> is not through conventional steric effects at the binding site but through reduced flexibility and changes in energy fluctuation pathways through the protein that influence its function. This is a phenomenon we have called “resistance by allostery”

Structural Insights into a Unique Inhibitor Binding Pocket in Kinesin Spindle Protein

Human kinesin Eg5 is a target for drug development in cancer chemotherapy with compounds in phase II clinical trials. These agents bind to a well-characterized allosteric pocket involving the loop L5 region, a structural element in kinesin-5 family members thought to provide inhibitor specificity. Using X-ray crystallography, kinetic, and biophysical methods, we have identified and characterized a distinct allosteric pocket in Eg5 able to bind inhibitors with nanomolar <i>K</i>_d. This pocket is formed by key structural elements thought to be pivotal for force generation in kinesins and may represent a novel site for therapeutic intervention in this increasingly well-validated drug target

Triphenylbutanamines: Kinesin Spindle Protein Inhibitors with in Vivo Antitumor Activity

The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified <i>S</i>-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold. This new generation systematically improves on potency: the most potent <i>C</i>-trityl analogues exhibit <i>K</i>_i^app ≤ 10 nM and GI₅₀ ≈ 50 nM, comparable to results from the phase II clinical benchmark ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as <i>S</i>-trityl analogues at an allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude mice xenograft studies

New insights into complex formation by SARS-CoV-2 nsp10 and nsp14

SARS-CoV-2 non-structural protein 10 (nsp10) is essential for the stimulation of enzymatic activities of nsp14 and nsp16, acting as both an activator and scaffolding protein. Nsp14 is a bifunctional enzyme with the N-terminus containing a 3′-5′ exoribonuclease (ExoN) domain that allows the excision of nucleotide mismatches at the virus RNA 3’-end, and a C-terminal N7-methyltransferase (N7-MTase) domain. Nsp10 is required for stimulating both ExoN proofreading and the nsp16 2’-O-methyltransferase activities. This makes nsp10 a central player in both viral resistance to nucleoside-based drugs and the RNA cap methylation machinery that helps the virus evade innate immunity. We characterised the interactions between full-length nsp10 (139 residues), N- and C-termini truncated nsp10 (residues 10-133), and nsp10 with a C-terminal truncation (residues 1-133) with nsp14 using microscale thermophoresis, multi-detection SEC, and hydrogen-deuterium (H/D) exchange mass spectrometry. We describe the functional role of the C-terminal region of nsp10 for binding to nsp14 and show that full N- and C-termini of nsp10 are important for optimal binding. In addition, our H/D exchange experiments suggest an intermediary interaction of nsp10 with the N7-MTase domain of nsp14. In summary, our results suggest intermediary steps in the process of association or dissociation of the nsp10–nsp14 complex, involving contacts between the two proteins in regions not identifiable by X-ray crystallography alone.</p

Ligand- and structure-based <i>in silico</i> studies to identify kinesin spindle protein (KSP) inhibitors as potential anticancer agents

<p>Kinesin spindle protein (KSP) belongs to the kinesin superfamily of microtubule-based motor proteins. KSP is responsible for the establishment of the bipolar mitotic spindle which mediates cell division. Inhibition of KSP expedites the blockade of the normal cell cycle during mitosis through the generation of monoastral MT arrays that finally cause apoptotic cell death. As KSP is highly expressed in proliferating/cancer cells, it has gained considerable attention as a potential drug target for cancer chemotherapy. Therefore, this study envisaged to design novel KSP inhibitors by employing computational techniques/tools such as pharmacophore modelling, virtual database screening, molecular docking and molecular dynamics. Initially, the pharmacophore models were generated from the data-set of highly potent KSP inhibitors and the pharmacophore models were validated against <i>in house</i> test set ligands. The validated pharmacophore model was then taken for database screening (Maybridge and ChemBridge) to yield hits, which were further filtered for their drug-likeliness. The potential hits retrieved from virtual database screening were docked using CDOCKER to identify the ligand binding landscape. The top-ranked hits obtained from molecular docking were progressed to molecular dynamics (AMBER) simulations to deduce the ligand binding affinity. This study identified MB-41570 and CB-10358 as potential hits and evaluated these experimentally using <i>in vitro</i> KSP ATPase inhibition assays.</p