Structure-based inhibitor design and repurposing clinical drugs to target SARS-CoV-2 proteases

SARS-CoV-2, the coronavirus responsible for the current COVID-19 pandemic, encodes two proteases, 3CLpro and PLpro, two of the main antiviral research targets. Here we provide an overview of the structures and functions of 3CLpro and PLpro and examine strategies of structure-based drug designing and drug repurposing against these proteases. Rational structure-based drug design enables the generation of potent and target-specific antivirals. Drug repurposing offers an attractive prospect with an accelerated turnaround. Thus far, several protease inhibitors have been identified, and some candidates are undergoing trials that may well prove to be effective antivirals against SARS-CoV-2.

Structures of the human peroxisomal fatty acid transporter ABCD1 in a lipid environment

The peroxisomal very long chain fatty acid (VLCFA) transporter ABCD1 is central to fatty acid catabolism and lipid biosynthesis. Its dysfunction underlies toxic cytosolic accumulation of VLCFAs, progressive demyelination, and neurological impairments including X-linked adrenoleukodystrophy (X-ALD). We present cryo-EM structures of ABCD1 in phospholipid nanodiscs in a nucleotide bound conformation open to the peroxisomal lumen and an inward facing conformation open to the cytosol at up to 3.5 Å resolution, revealing details of its transmembrane cavity and ATP dependent conformational spectrum. We identify features distinguishing ABCD1 from its closest homologs and show that coenzyme A (CoA) esters of VLCFAs modulate ABCD1 activity in a species dependent manner. Our data suggest a transport mechanism where the CoA moieties of VLCFA-CoAs enter the hydrophilic transmembrane domain while the acyl chains extend out into the surrounding membrane bilayer. The structures help rationalize disease causing mutations and may aid ABCD1 targeted structure-based drug design.

Fragment based drug design

Rational approaches towards drug development have emerged as one of the most promising ways among the tedious conventional procedures with the aim of redefining the drug discovery process. The need of current medical system is demanding a much precise, faster and reliable approaches in parallel to faster growing technology for development of drugs with more intrinsic action and fewer side effects. Systematic and well-defined diagnostic studies have revealed the specific causes of disease and related targets for drug development. Designing a drug as per the specific target, studying it in-silico prior to its development has been proved as an added benefit to the studies. Many approaches like structure based drug design, fragment based drug design and ligand based drug design are been in practice for the drug discovery and development with the similar fundamental theory. Fragment based drug design utilizes a library of fragments designed specifically for the concerned target and these fragments are studied further before screening with virtual methods as well as with biophysical methods. The process follows a well-defined pathway which moulds a fragment into a perfect drug candidate. In this chapter we have tried to cover all the basic aspects of fragment based drug design and related technologies.

Recent Advances in Structure, Function, and Pharmacology of Class A Lipid GPCRs: Opportunities and Challenges for Drug Discovery

Great progress has been made over the past decade in understanding the structural, functional, and pharmacological diversity of lipid GPCRs. From the first determination of the crystal structure of bovine rhodopsin in 2000, much progress has been made in the field of GPCR structural biology. The extraordinary progress in structural biology and pharmacology of GPCRs, coupled with rapid advances in computational approaches to study receptor dynamics and receptor-ligand interactions, has broadened our comprehension of the structural and functional facets of the receptor family members and has helped usher in a modern age of structure-based drug design and development. First, we provide a primer on lipid mediators and lipid GPCRs and their role in physiology and diseases as well as their value as drug targets. Second, we summarize the current advancements in the understanding of structural features of lipid GPCRs, such as the structural variation of their extracellular domains, diversity of their orthosteric and allosteric ligand binding sites, and molecular mechanisms of ligand binding. Third, we close by collating the emerging paradigms and opportunities in targeting lipid GPCRs, including a brief discussion on current strategies, challenges, and the future outlook.

Cryo-EM Structure-guided Selection of Computed Ligand Poses to Enhance Potency in MTA-synergic Inhibition of Human Protein Arginine Methyltransferase 5

The potential of using cryo-electron microscopic (cryo-EM) structures of 2.5-4.0 Å resolutions for structure-based drug design was proposed recently, but is yet to be materialized. Here we show that a 3.1 Å cryo-EM structure of protein arginine methyltransferase 5 (PRMT5) is sufficient to guide the selection of computed poses of a bound inhibitor and its redesign for much higher potency. PRMT5 is an oncogenic target and its multiple inhibitors are in clinical trials for various cancer types. However, all these PRMT5 inhibitors manifest negative cooperativity with a metabolic co-factor analog --- 2-methylthioadenosine (MTA), which is accumulated substantially in cancer patients carrying defective MTA phosphorylase (MTAP). To achieve MTA-synergetic inhibition, we obtained a pharmacophore from virtual screen and synthesized a specific inhibitor (11-2F). Cryo-EM structures of the 11-2F/MTA-bound human PRMT5: MEP50 complex and its apo form together showed that the inhibitor binding in the catalytic pocket causes a shift of the cofactor-binding site by 1.5 – 2.0 Å, disfavoring cofactor-binding and resulting in positive cooperativity between 11-2F and MTA. Coarse-grained and full-atomistic MD simulations of the ligands in their binding pockets were performed to compare computed poses of 11-2F and its redesigned analogs. Three new analogs were predicted to have much better potency. One of them, after synthesis, was ~4 fold more efficient in PRMT5 inhibition in the presence of MTA than 11-2F itself. Computational analysis also suggests strong subtype specificity of 11-2F among PRMTs. These data demonstrate the feasibility of using cryo-EM structures of near-atomic resolutions and computational analysis of ligand poses for better small molecule therapeutics.

Modeling Alternate Conformations with Alphafold2 via Modification of the Multiple Sequence Alignment

The unprecedented performance of Deepmind's Alphafold2 in predicting protein structure in CASP XIV and the creation of a database of structures for multiple proteomes is reshaping structural biology. Moreover, the availability of Alphafold2's architecture and code has stimulated a number of questions on how to harness the capabilities of this remarkable tool. A question of central importance is whether Alphafold2's architecture is amenable to predict the intrinsic conformational heterogeneity of proteins. A general approach presented here builds on a simple manipulation of the multiple sequence alignment, via in silico mutagenesis, and subsequent modeling by Alphafold2. The approach is based in the concept that the multiple sequence alignment encodes for the structural heterogeneity, thus its rational manipulation will enable Alphafold2 to sample alternate conformations and potentially structural alterations due to point mutations. This modeling pipeline is benchmarked against canonical examples of protein conformational flexibility and applied to interrogate the conformational landscape of membrane proteins. This work broadens the applicability of Alphafold2 by generating multiple protein conformations to be tested biologically, biochemically, biophysically, and for use in structure-based drug design.

Applications of Cryo-EM in small molecule and biologics drug design

Electron cryo-microscopy (cryo-EM) is a powerful technique for the structural characterization of biological macromolecules, enabling high-resolution analysis of targets once inaccessible to structural interrogation. In recent years, pharmaceutical companies have begun to utilize cryo-EM for structure-based drug design. Structural analysis of integral membrane proteins, which comprise a large proportion of druggable targets and pose particular challenges for X-ray crystallography, by cryo-EM has enabled insights into important drug target families such as G protein-coupled receptors (GPCRs), ion channels, and solute carrier (SLCs) proteins. Structural characterization of biologics, such as vaccines, viral vectors, and gene therapy agents, has also become significantly more tractable. As a result, cryo-EM has begun to make major impacts in bringing critical therapeutics to market. In this review, we discuss recent instructive examples of impacts from cryo-EM in therapeutics design, focusing largely on its implementation at Pfizer. We also discuss the opportunities afforded by emerging technological advances in cryo-EM, and the prospects for future development of the technique.