Rapid Covalent-Probe Discovery by Electrophile-Fragment Screening

Covalent probes can display unmatched potency, selectivity, and duration of action; however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening, but such electrophilic fragments were considered nonselective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against 10 cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. In contrast, we found hits for most targets. Combining our approach with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2 and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery

Biochemical, Structural, And Drug Design Studies Of Norovirus And Zika Virus Proteases

Noroviruses, which are the leading cause of acute gastroenteritis, cause an estimated 677 million infections and 213,000 deaths each year worldwide. Noroviruses are classified into seven genogroups (GI-GVII); GI, GII, and GIV have been shown to be infectious in humans. However, GII noroviruses cause the majority of outbreaks (89%). No pharmacologic treatment or vaccine currently exists to treat or prevent norovirus infections. Recently, the development of a norovirus replicon system, a murine model of norovirus infection, and the development of a biochemical protease assay have allowed for the design and development of norovirus inhibitors. However, the replicon and biochemical assay were developed with GI noroviruses. In this work, we have developed a system to design, evaluate, and develop inhibitors against GII noroviruses, which are responsible for the majority of outbreaks. Using molecular dynamics simulations and other computational tools, we have shown that GII norovirus proteases behave comparably in solution with GI norovirus proteases in terms of protein flexibility as well as binding site solvent exposure, druggability, hydrophobicity, and volume. Therefore, we propose that protease inhibitors designed against either GI or GII norovirus proteases would be cross-reactive with the other genogroup – a broad-spectrum norovirus protease inhibitor is likely feasible. In addition, we have developed a fluorescence based biochemical assay to design and evaluate protease inhibitors against GII norovirus proteases, specifically a GII.4 norovirus protease. Using the biochemical assay and computational techniques, we also show that peptidomimetic inhibitors containing and aldehyde as an electrophilic warhead inhibit a GII.4 norovirus protease more potently than peptidomimetic inhibitors which contain an α,β-unsaturated ethyl ester Michael acceptor moiety as an electrophilic warhead. Additionally, this work also explored the Zika virus (ZIKV) NS2B-NS3 protease as a potential drug target. ZIKV, an emerging flavivirus, was first discovered in 1947 but only recently caused an infectious outbreak of international concern in the Americas in 2015-2016. ZIKV diverged into two distinct lineages: African and Asian. The recent outbreak in the Americas, which has caused a total of 171,553 confirmed infections over 48 countries, is associated with the Asian ZIKV lineage. Previously thought to be a mild infection, the recent outbreak, which is spread by the Aedes spp. mosquitoes, sexual-transmission, as well as vertical transmission from mother to fetus, is associated with more severe complications, the main concern being the rise in microcephaly cases. Depending on when ZIKV was contracted, 10-15% of pregnancies with laboratory confirmed ZIKV infection result in birth defects. No vaccine or pharmacologic therapy yet exists to prevent or treat ZIKV infections. However, similar to other flaviviruses, the NS2B/NS3 protease has been proposed as a potential drug target. The NS2B protein, a membrane bound protein, exists acts as a cofactor for the NS3 protease during viral polyprotein cleavage. The structure of the ZIKV NS2B/NS3 protease was recently solved by X-ray crystallography, and a fluorescence based biochemical assay has been proposed in order to design, evaluate, and develop NS2B/NS3 protease inhibitors. The X-ray crystal structure and biochemical assay were developed with the NS2B cofactor covalently linked to the NS3 protease. In this work, we created the linked NS2B/NS3 protease, as well as unlinked NS2B/NS3 protease for comparison. The unlinked NS2B/NS3 protease is roughly five-times more active than the linked NS2B/NS3 protease. Molecular dynamics simulations suggest that covalently linking the NS2B cofactor to the NS3 protease may reduce the substrate binding region flexibility as well as sterically hinder substrate binding. Therefore, we propose that unlinked NS2B/NS3 protease be used to design, evaluate, and develop ZIKV protease inhibitors. This work has resulted in valuable tools and structural insights that can aid the design and development of both norovirus and Zika virus protease inhibitors. In addition, the techniques described can be used to study other proteins, further understand protease behavior, and design new compounds

A new covalent PIN1 inhibitor selectively targets cancer cells by a dual mechanism of action

openIn the last decades targeted drugs have improved cancer treatment, but revealed to be ineffective mainly in the treatment of solid tumors, largely because of tumor heterogeneity, activation of redundant pathways, and drug resistance. A common and central signal transduction mechanism in many oncogenic pathways is the phosphorylation of proteins at serine or threonine residues followed by proline (S/T-P). Importantly, the phospho-S/T-P motifs of these proteins are recognized by the peptidyl-prolyl cis/trans isomerase (PPIase) PIN1, which catalyzes the cis-trans or trans-cis conformational change around the S-P or T-P bond. Among PPIases, PIN1 is the only enzyme able to efficiently bind proteins containing phosphorylated S/T-P motifs. As a consequence, the phosphorylation dependent prolyl-isomerase PIN1 acts as a critical modifier of multiple signaling pathways. It is overexpressed in the majority of cancers and its activity strongly contributes to tumor initiation and progression. Conversely, inactivation of PIN1 function curbs tumor growth and cancer stem cell expansion, restores chemosensitivity and blocks metastatic spread, thus providing the rationale for a therapeutic strategy based on PIN1 inhibition. Notwithstanding, potent PIN1 inhibitors are still missing from the arsenal of anti-cancer drugs. By a mechanism-based screening we have identified a novel covalent PIN1 inhibitor, KPT-6566, able to selectively inhibit PIN1 among other prolyl-isomerases, and target it for degradation. We demonstrate that KPT-6566 covalently binds to the catalytic site of PIN1. This interaction results in the release of a quinine-mimicking drug that generates reactive oxygen species and DNA damage inducing cell death specifically in cancer cells. Accordingly, KPT-6566 treatment impairs PIN1-dependent cancer phenotypes in vitro and growth of lung metastasis in vivo.BIOMEDICINA MOLECOLAREembargoed_20180509Campaner, ElenaCampaner, Elen

Structure-Based Virtual Screening Approach for Discovery of Covalently Bound Ligands

We present a fast and effective covalent docking approach suitable for large-scale virtual screening (VS). We applied this method to four targets (HCV NS3 protease, Cathepsin K, EGFR, and XPO1) with known crystal structures and known covalent inhibitors. We implemented a customized “VS mode” of the Schrödinger Covalent Docking algorithm (CovDock), which we refer to as CovDock-VS. Known actives and target-specific sets of decoys were docked to selected X-ray structures, and poses were filtered based on noncovalent protein–ligand interactions known to be important for activity. We were able to retrieve 71%, 72%, and 77% of the known actives for Cathepsin K, HCV NS3 protease, and EGFR within 5% of the decoy library, respectively. With the more challenging XPO1 target, where no specific interactions with the protein could be used for postprocessing of the docking results, we were able to retrieve 95% of the actives within 30% of the decoy library and achieved an early enrichment factor (EF1%) of 33. The poses of the known actives bound to existing crystal structures of 4 targets were predicted with an average RMSD of 1.9 Å. To the best of our knowledge, CovDock-VS is the first fully automated tool for efficient virtual screening of covalent inhibitors. Importantly, CovDock-VS can handle multiple chemical reactions within the same library, only requiring a generic SMARTS-based predefinition of the reaction. CovDock-VS provides a fast and accurate way of differentiating actives from decoys without significantly deteriorating the accuracy of the predicted poses for covalent protein–ligand complexes. Therefore, we propose CovDock-VS as an efficient structure-based virtual screening method for discovery of novel and diverse covalent ligands

Study of the mechanism of action of bioactive plants tarpenoids

2014-2015Natural products are small-molecule secondary metabolites displaying considerable structural complexity and “privileged scaffolds”. They are able to bind several endogenous targets eliciting biological effects as chemical weapons or to convey information from one organism to another. Nowadays, medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets. Therefore, the primary purpose of this PhD thesis has been a comprehensive characterization of the interactome profile and then the molecular mechanism of action of bioactive natural molecules. Achieving this in an effective, unbiased and efficient manner subsists as a significant challenge for the new era in drug discovery and optimization. Indeed, the full understanding of the mechanism of action of natural molecules could lead to a number of advantages: first of all, exploit their full therapeutic potential, the identification of side effects or toxicity, or the ability to set up target-based assays and to allow structure activity relationships studies to guide medicinal chemistry efforts towards lead optimization. In my research project, the attention was paid on ent-kaurane diterpenes, a class of natural terpenoids with a great structural variability and a wide spectrum of biological activities. Firstly, I focused on the determination of the interactome of a semi synthetic compound 15-ketoatractyligenin methyl ester. This compound has been previously reported to possess high antiproliferative activity against several solid tumor-derived cell lines. In this regard, I decided to investigate the mechanism of action of this actratylignin derivative researching first of all its molecular targets, responsible for the biological activity. In order to achieve this goal, I used a chemical proteomic approach first. This study led to the identification of PPARγ as the main cellular partner of this compound; achieved results were supported and validated through different biological assays. Subsequently, I studied another diterpene: oridonin. This molecule has been shown to have multiple biological activities. Among them, the anticancer activity has been repeatedly reported by many research groups. With the aim of expanding and validate our knowledge about this molecule, also seen the limitations of the fishing for partners method, I decided to use two orthogonal compound-centric proteomics approaches to define the possible protein target(s) of oridonin. Using this strategy HSP70 and nucleolin were identified. Therefore, several in vitro and in cell tests have been performed to validate the interaction of oridonin with these proteins, and to evaluate its effect on their activity. Some of these tests were developed and optimized during my period of research abroad at the Massachusset General Hospital- Center for System Biology -Harvard Medical School; in that twelve months period I expanded my knowledge into the techniques useful for the study of the mechanism of action of a small molecule, also applying experimental methods complementary to proteomics and focusing on the use of high-resolution intravital microscopy imaging for drug pharmacology. [edited by Author]XIV n.s