Virtual Combinatorial Chemistry and Pharmacological Screening: A Short Guide to Drug Design.

Traditionally, drug development involved the individual synthesis and biological evaluation of hundreds to thousands of compounds with the intention of highlighting their biological activity, selectivity, and bioavailability, as well as their low toxicity. On average, this process of new drug development involved, in addition to high economic costs, a period of several years before hopefully finding a drug with suitable characteristics to drive its commercialization. Therefore, the chemical synthesis of new compounds became the limiting step in the process of searching for or optimizing leads for new drug development. This need for large chemical libraries led to the birth of high-throughput synthesis methods and combinatorial chemistry. Virtual combinatorial chemistry is based on the same principle as real chemistry-many different compounds can be generated from a few building blocks at once. The difference lies in its speed, as millions of compounds can be produced in a few seconds. On the other hand, many virtual screening methods, such as QSAR (Quantitative Sturcture-Activity Relationship), pharmacophore models, and molecular docking, have been developed to study these libraries. These models allow for the selection of molecules to be synthesized and tested with a high probability of success. The virtual combinatorial chemistry-virtual screening tandem has become a fundamental tool in the process of searching for and developing a drug, as it allows the process to be accelerated with extraordinary economic savings.

A high-throughput pipeline for design and selection of peptides targeting the SARS-Cov-2 Spike protein.

Rapid design, screening, and characterization of biorecognition elements (BREs) is essential for the development of diagnostic tests and antiviral therapeutics needed to combat the spread of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address this need, we developed a high-throughput pipeline combining in silico design of a peptide library specific for SARS-CoV-2 spike (S) protein and microarray screening to identify binding sequences. Our optimized microarray platform allowed the simultaneous screening of ~ 2.5 k peptides and rapid identification of binding sequences resulting in selection of four peptides with nanomolar affinity to the SARS-CoV-2 S protein. Finally, we demonstrated the successful integration of one of the top peptides into an electrochemical sensor with a clinically relevant limit of detection for S protein in spiked saliva. Our results demonstrate the utility of this novel pipeline for the selection of peptide BREs in response to the SARS-CoV-2 pandemic, and the broader application of such a platform in response to future viral threats.

Targeting Ribosomal Frameshifting as an Antiviral Strategy: From HIV-1 to SARS-CoV-2.

Treatment of HIV-1 has largely involved targeting viral enzymes using a cocktail of inhibitors. However, resistance to these inhibitors and toxicity in the long term have pushed the field to identify new therapeutic targets. To that end, -1 programmed ribosomal frameshifting (-1 PRF) has gained attention as a potential node for therapeutic intervention. In this process, a ribosome moves one nucleotide backward in the course of translating a mRNA, revealing a new reading frame for protein synthesis. In HIV-1, -1 PRF allows the virus to regulate the ratios of enzymatic and structural proteins as needed for correct viral particle assembly. Two RNA structural elements are central to -1 PRF in HIV: a slippery sequence and a highly conserved stable hairpin called the HIV-1 frameshifting stimulatory signal (FSS). Dysregulation of -1 PRF is deleterious for the virus. Thus, -1 PRF is an attractive target for new antiviral development. It is important to note that HIV-1 is not the only virus exploiting -1 PRF for regulating production of its proteins. Coronaviruses, including the COVID-19 pandemic virus SARS-CoV-2, also rely on -1 PRF. In SARS-CoV-2 and other coronaviruses, -1 PRF is required for synthesis of RNA-dependent RNA polymerase and several other nonstructural proteins. Coronaviruses employ a more complex RNA structural element for regulating -1 PRF called a pseudoknot. The purpose of this Account is primarily to review the development of molecules targeting HIV-1 -1 PRF. These approaches are case studies illustrating how the entire pipeline from screening to the generation of high-affinity leads might be implemented. We consider both target-based and function-based screening, with a particular focus on our group's approach beginning with a resin-bound dynamic combinatorial library (RBDCL) screen. We then used rational design approaches to optimize binding affinity, selectivity, and cellular bioavailability. Our tactic is, to the best of our knowledge, the only study resulting in compounds that bind specifically to the HIV-1 FSS RNA and reduce infectivity of laboratory and drug-resistant strains of HIV-1 in human cells. Lessons learned from strategies targeting -1 PRF HIV-1 might provide solutions in the development of antivirals in areas of unmet medical need. This includes the development of new frameshift-altering therapies for SARS-CoV-2, approaches to which are very recently beginning to appear.

De novo design, retrosynthetic analysis and combinatorial synthesis of a hybrid antiviral (VTAR-01) to inhibit the interaction of SARS-CoV2 spike glycoprotein with human angiotensin-converting enzyme 2.

SARS-like coronavirus (SARS-CoV2) has emerged as a global threat to humankind and is rapidly spreading. The infectivity, pathogenesis and infection of this virus are dependent on the interaction of SARS-CoV2 spike protein with human angiotensin converting enzyme 2 (hACE2). Spike protein contains a receptor-binding domain (RBD) that recognizes hACE-2. In the present study, we are reporting a de novo designed novel hybrid antiviral 'VTAR-01' molecule that binds at the interface of RBD-hACE2 interaction. A series of antiviral molecules were tested for binding at the interface of RBD-hACE2 interaction. In silico screening, molecular mechanics and molecular dynamics simulation (MDS) analysis suggest ribavirin, ascorbate, lopinavir and hydroxychloroquine have strong interaction at the RBD-hACE2 interface. These four molecules were used for de novo fragment-based antiviral design. De novo designing, docking and MDS analysis identified a 'VTAR' hybrid molecule that has better interaction with this interface than all of the antivirals used to design it. We have further used retrosynthetic analysis and combinatorial synthesis to design 100 variants of VTAR molecules. Retrosynthetic analysis and combinatorial synthesis, along with docking and MDS, identified that VTAR-01 interacts with the interface of the RBD-ACE2 complex. MDS analysis confirmed its interaction with the RBD-ACE2 interface by involving Glu35 and Lys353 of ACE2, as well as Gln493 and Ser494 of RBD. Interaction of spike protein with ACE2 is essential for pathogenesis and infection of this virus; hence, this in silico designed hybrid antiviral molecule (VTAR-01) that binds at the interface of RBD-hACE2 may be further developed to control the infection of SARS-CoV2.

SARS-CoV-2 Mpro inhibitors and activity-based probes for patient-sample imaging.

In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed. Currently, there is no effective antiviral treatment for COVID-19. To address this emerging problem, we focused on the SARS-CoV-2 main protease that constitutes one of the most attractive antiviral drug targets. We have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases. On the basis of these findings, we designed and synthesized a potent SARS-CoV-2 inhibitor (Ac-Abu-DTyr-Leu-Gln-VS, half-maximal effective concentration of 3.7 µM) and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. We visualized active SARS-CoV-2 Mpro in nasopharyngeal epithelial cells of patients suffering from COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents and/or diagnostic tests.