Overcoming Chemical, Biological, and Computational Challenges in the Development of Inhibitors Targeting Protein-Protein Interactions.

Protein-protein interactions (PPIs) underlie the majority of biological processes, signaling, and disease. Approaches to modulate PPIs with small molecules have therefore attracted increasing interest over the past decade. However, there are a number of challenges inherent in developing small-molecule PPI inhibitors that have prevented these approaches from reaching their full potential. From target validation to small-molecule screening and lead optimization, identifying therapeutically relevant PPIs that can be successfully modulated by small molecules is not a simple task. Following the recent review by Arkin et al., which summarized the lessons learnt from prior successes, we focus in this article on the specific challenges of developing PPI inhibitors and detail the recent advances in chemistry, biology, and computation that facilitate overcoming them. We conclude by providing a perspective on the field and outlining four innovations that we see as key enabling steps for successful development of small-molecule inhibitors targeting PPIs.Work in DRS’s laboratory is supported by the the European Union, Engineering and Physical Sciences Research Council, Biotechnology and Biological Sciences Research Council, Medical Research Council and Wellcome Trust. Work in ARV’s laboratory is supported by the Medical Research Council and Wellcome Trust. Work in DJH's laboratory is supported by the Medical Research Council under grant ML/L007266/1. All calculations were performed using the Darwin Supercomputer of the University of Cambridge High Performance Computing Service (http://www.hpc.cam.ac.uk/) provided by Dell Inc. using Strategic Research Infrastructure Funding from the Higher Education Funding Council for England and were funded by the EPSRC under grants EP/F032773/1 and EP/J017639/1. GJM and ARV are affiliated with PhoreMost Ltd, Cambridge. We thank Alicia Higueruelo and John Skidmore for helpful discussions.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.chembiol.2015.04.01

IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD

Computational approaches guiding for the design and optimization of novel chemo-types endowed with F508del-CFTR modulator ability

To date, monotherapy with VX-809 (Lumacaftor) or VX-770 (Ivacaftor) has not resulted in obvious clinical benefits for CF patients, while their combination regimen has provided positive results, stabilizing disease progress. Consequently, therapy combined with dual modulators or triple combination represents today the most promising prospect for developing new therapies. In this context, the research group in which I have been carrying out this thesis has dealt with rational design and computational studies of CFTR modulators during the past few years. The information obtained from our previous studies allowed us to proceed with the rational design and to predict the possible corrective activity of a new series of compounds with an aminoarylthiazole structure (AAT)1.,165. The previously proposed studies' reliability was supported by biological studies carried out on the newly synthesized molecules in collaboration with the research group led by Dr. Nicoletta Pedemonte (Istituto Giannina Gaslini, Genoa), verifying the corrective activity for F508del-CFTR of the newly designed derivatives. About the computational approaches so far applied, a QSAR model has been developed on the correctors available in literature guiding the following design and synthesis of hybrids compounds. This ligand-based method was used to overcome the paucity of information regarding a single and specific mechanism of action responsible for the corrective activity of VX-809. Indeed, as described in the literature, several hypotheses suggest multiple sites on the CFTR protein to which VX-809 could bind, first of all, the NBD1 domain. This thesis deepened the structure-based approach concerning various correctors described in the literature, including the hybrids developed by the present research group. In this context, experimental but partial data of the NBD1 domain of F508del-CFTR (PDB code: 4WZ6) were considered to perform molecular docking simulations of the compounds mentioned above. This research has been completed by molecular docking calculations performed on a whole model of the F508del-CFTR protein, which has been built in silico by our research group. Unlike what occurs for CFTR correctors, applying structure-based methods in the rational design of potentiators appears to be a more straightforward strategy since the experimental data concerning the binding mode of the VX-770 potentiator has recently become available (PDB code = 6O2P) and GLP1837 (PDB code = 6O1V). Starting from these assumptions, in this thesis, several libraries of compounds, described in the literature as CFTR potentiators, such as indoles, pyrazolquinolines, thienopyranes, cyanoquinolines, and AAT, have been studied to perform molecular docking studies and QSAR analysis activities. These approaches allowed us to obtain information to guide the rational design and future synthesis of new CFTR modulators. The research activity's further goal was to apply - in parallel to the studies just mentioned - ligand-based drug design analysis, using classical QSAR type analysis. This approach made it possible to overcome any limitation related to uniquely examining a single possible target for CFTR modulators and focusing on chemical scaffolds known today as correctors or potentiators

Discovery of OJT008 as a Novel Inhibitor of Mycobacterium tuberculosis

Despite recent progress in the diagnosis of Tuberculosis (TB), the chemotherapeutic management of TB is still challenging. Mycobacterium tuberculosis (Mtb) is the etiological agent of TB, and TB is classified as the 13th leading cause of death globally [WHO 2021]. 558,000 people were reported to develop multi-drug resistant TB globally [WHO 2020]. Our research focuses on targeting Methionine Aminopeptidase (MetAP), an essential protein for the viability of Mtb. MetAP is a metalloprotease that catalyzes the removal of N-terminal methionine (NME) during translation of protein [Giglione et al., 2003]. This essential role of MetAPs makes this enzyme an auspicious target for the development of novel therapeutic agents for the treatment of TB. Mtb possesses two MetAP1 isoforms: MtMetAP1a and MtMetAP1c, which are vital for Mtb viability, hence a promising chemotherapeutic target for Mtb infection [Zhang et al., 2009; Olaleye et al., 2010; Griffin et al., 2011; Vanunu et al., 2019]. In our study, we cloned, overexpressed recombinant MtMetAP1c, and investigated the in vitro inhibitory effect of OJT008 on cobalt and nickel ion activated MtMetAP1c. The compound’s potency against replicating and multidrug-resistant (MDR) Mtb strains was also investigated. The induction of the overexpressed recombinant MtMetAP1c was optimized at hours with a final concentration of 1mM Isopropyl β-D-1-thiogalactopyranoside. The average yield for MtMetAP1c was 4.65 mg/L of Escherichia coli culture. A preliminary MtMetAP1c metal dependency screen showed optimum activation with nickel and cobalt ions at 100µM. The half-maximal inhibitory concentration (IC50) values of OJT008 against MtMetAP1c activated with CoCl2 and NiCl2 were in the micromolar range. Our in silico study showed OJT008 strongly binds to both metal activated MtMetAP1c, as evidenced by strong molecular interactions and higher binding score thereby corroborating our result. Thus, validating the pharmacophore’s metal specificity. The potency of OJT008 against both active and multidrug-resistant (MDR) Mtb was in the low micromolar concentrations, correlating well with our biochemical data on MtMetAP1c inhibition. These results suggest that OJT008 is a potential lead compound for the pre-clinical development of novel small molecules for the therapeutic management of TB

Evaluation of Aminopyrazole Analogs as Cyclin-Dependent Kinase Inhibitors for Colorectal Cancer Therapy

Colorectal cancer (CRC) remains one of the leading causes of cancer related deaths in the United States. Currently, there are limited therapeutic options for CRC patients, none of which focus on the cell signaling mechanisms controlled by members of the cyclin dependent kinase (CDK) family. CDK5 has been implicated in a variety of cancers, and most recently as a tumor promoter in CRC. As such, we evaluated a compound developed by Pfizer, CP-668863 (a.k.a. 20-223), that inhibits CDK5 in neurodegenerative disorders. In our CRC xenograft model, 20-223 reduced tumor growth and tumor weight, indicating its value as a potential anti-CRC agent. We subjected 20-223 to a series of cell-free and cell-based studies to understand the mechanism of its anti-tumor effects. Profiling the CDK family revealed that 20-223 was most potent against CDK2 and CDK5 in cell-free and cell-based systems. The clinically used CDK inhibitor AT7519 and 20-223 share the aminopyrazole core. 20-223 was comparable, or in some cases better, than clinically used AT7519, proving it to be a suitable lead compound. Next we utilized the new PRoteolysis TArgeting Chimera (PROTAC) strategy to develop CDK5 degraders. Synthesis and evaluation revealed that the heterobifunctional aminopyrazole-based PROTAC capable of cereblon-mediated proteasomal degradation targeted CDK9 while sparing CDK2 and CDK5. While the degrader (3) did in fact bind to CDK5 and inhibit its kinase activity, it was unable to trigger its degradation, likely due to differentially exposed lysine residues. This is the first report of a PROTAC capable of degrading a member of the oncogenic CDK family. Overall, these studies demonstrate that inhibition of CDK5 is a promising therapeutic strategy and warrants further evaluation. 20-223 is a favorable lead compound for CRC therapy as it exhibits anti-cancer activity both in vitro and in vivo. Additionally, the PROTAC strategy can be applied to develop selective CDK degraders

Unconventional approaches to kinase inhibition : covalent inhibitors and docking site inhibitors of mitogen-activated protein kinases

The body of work that follows is a description of biochemical screening methods to identify non-ATP competitive and covalent ERK inhibitors, as well as assay design for characterizing covalent inhibitors of JNK in an isoform-specific manner. A covalent ERK inhibitor is presented, as well as a novel class of non-covalent ERK docking site inhibitors. An assay to elucidate the kinetic parameters of covalent JNK inhibitors is described, and the kinetic parameters for the inhibitor JNK-IN-8 are illustrated as a case study. Additionally, a series of JNK-IN-8 analogues were tested in a PyVMT breast cancer cell model for their ability to preferentially inhibit either JNK1 or JNK2. The rationale behind targeting MAPKs at docking sites and focusing on covalent inhibitors is centered on several key elements. First, the proximity-based mechanism of MAPK catalysis depends on docking site interactions to direct substrate consensus sequences to the vicinity of the MAPK active site. MAPK activators, phosphatases, scaffolding proteins, and other molecules also engage docking sites. Therefore, targeting docking sites with inhibitors can not only directly or indirectly block substrate phosphorylation or enzyme activation, but the mechanisms of catalysis themselves can be altered. Secondly, the signal dynamics of MAPKs, such as their switch-like behavior, intra-pathway feedback, and inter-pathway crosstalk makes complete inhibition of MAPKs necessary to stop signaling in a therapeutic context. Covalent, irreversible MAPK inhibitors can induce complete inhibition by being effectively immune to substrate competition, and by forcing recovery of signaling to be fully dependent on either MAPK mutations, protein synthesis, or pathway bypass. Targeting a MAPK at multiple interaction sites can further promote a full blockade of signaling. The collective goal of the work presented in this dissertation is to contribute to the breadth of targeted kinase inhibitors that can be used against cancers, such as BRAF-V600E melanoma. These inhibitors and methods of inhibitor development can be used as tools to probe the functionality of individual protein-protein interaction sites on ERK and JNK, and the principles described here can be adapted to apply to other kinases and binding sites as needed in future studies.Biomedical Engineerin

In Silico Design and Selection of CD44 Antagonists:implementation of computational methodologies in drug discovery and design

Drug discovery (DD) is a process that aims to identify drug candidates through a thorough evaluation of the biological activity of small molecules or biomolecules. Computational strategies (CS) are now necessary tools for speeding up DD. Chapter 1 describes the use of CS throughout the DD process, from the early stages of drug design to the use of artificial intelligence for the de novo design of therapeutic molecules. Chapter 2 describes an in-silico workflow for identifying potential high-affinity CD44 antagonists, ranging from structural analysis of the target to the analysis of ligand-protein interactions and molecular dynamics (MD). In Chapter 3, we tested the shape-guided algorithm on a dataset of macrocycles, identifying the characteristics that need to be improved for the development of new tools for macrocycle sampling and design. In Chapter 4, we describe a detailed reverse docking protocol for identifying potential 4-hydroxycoumarin (4-HC) targets. The strategy described in this chapter is easily transferable to other compounds and protein datasets for overcoming bottlenecks in molecular docking protocols, particularly reverse docking approaches. Finally, Chapter 5 shows how computational methods and experimental results can be used to repurpose compounds as potential COVID-19 treatments. According to our findings, the HCV drug boceprevir could be clinically tested or used as a lead molecule to develop compounds that target COVID-19 or other coronaviral infections. These chapters, in summary, demonstrate the importance, application, limitations, and future of computational methods in the state-of-the-art drug design process

Computational Approaches: Drug Discovery and Design in Medicinal Chemistry and Bioinformatics

This book is a collection of original research articles in the field of computer-aided drug design. It reports the use of current and validated computational approaches applied to drug discovery as well as the development of new computational tools to identify new and more potent drugs

Evolution and Prevention of Antibiotic Resistance: Small Molecule Inhibitors of Bacterial Recombination Enzymes

Antibiotic resistant bacteria are an ever-increasing problem for the modern chemotherapy of bacterial infectious diseases. The loss of effective antibiotic therapies due to antibiotic resistance and the withering antibiotic pipeline are resulting in a reemergence in deaths from bacterial infections. New strategies are needed to combat pathogenic bacteria and in this context bacterial targets involved in the development of resistance are emerging an intriguing candidates for inhibition studies. Recent evidence suggests that bacterial stress response pathways (i.e., SOS and competence for transformation) are responsible for accelerated genetic changes that ultimately establish antibiotic resistance. Intervening in these pathways by small molecule inhibition of key recombination enzymes, RecA and EndA, would impact the DNA repair, SOS mutagenesis and recombination-based horizontal gene transfer activities of these enzymes and hinder the acquisition of antibiotic resistance. Bacteria having loss-of-function mutations in the recA gene are more sensitive to antibiotic treatment and develop resistance more slowly or not at all. In addition, endA-null strains of S. pneumoniae have diminished transformation efficiencies and are unable to acquire resistance-conferring DNA. Therefore, we believe chemotherapeutic agents that impart these bacterial phenotypes could act synergistically with currently prescribed antibiotics to prevent the accumulation of populations that are resistant to them. Towards this goal, we sought to identify properly designed inhibitors of RecA and EndA. High-throughput screening (HTS) is recognized as a powerful tool in drug discovery to identify target-specific lead compounds. We developed rational high-throughput screening programs to discover small-molecule inhibitors of RecA and EndA. Through these studies, we have identified novel chemical classes that specifically target RecA or EndA and demonstrate that these enzymes hold potential as novel targets in the treatment of bacterial infections.Doctor of Philosoph