Large-Scale Virtual Screening for the Discovery of SARS-CoV-2 Papain-like Protease (PLpro) Non-covalent Inhibitors.

The rapid global spread of the SARS-CoV-2 virus facilitated the development of novel direct-acting antiviral agents (DAAs). The papain-like protease (PLpro) has been proposed as one of the major SARS-CoV-2 targets for DAAs due to its dual role in processing viral proteins and facilitating the host's immune suppression. This dual role makes identifying small molecules that can effectively neutralize SARS-CoV-2 PLpro activity a high-priority task. However, PLpro drug discovery faces a significant challenge due to the high mobility and induced-fit effects in the protease's active site. Herein, we virtually screened the ZINC20 database with Deep Docking (DD) to identify prospective noncovalent PLpro binders and combined ultra-large consensus docking with two pharmacophore (ph4)-filtering strategies. The analysis of active compounds revealed their somewhat-limited diversity, likely attributed to the induced-fit nature of PLpro's active site in the crystal structures, and therefore, the use of rigid docking protocols poses inherited limitations. The top hits were assessed against recombinant viral proteins and live viruses, demonstrating desirable inhibitory activities. The best compound VPC-300195 (IC50: 15 µM) ranks among the top noncovalent PLpro inhibitors discovered through in silico methodologies. In the search for novel SARS-CoV-2 PLpro-specific chemotypes, the identified inhibitors could serve as diverse templates for the development of effective noncovalent PLpro inhibitors.

Development of fluorescent peptide substrates and assays for the key autophagy-initiating cysteine protease enzyme, ATG4B.

An efficient assay for monitoring the activity of the key autophagy-initiating enzyme ATG4B based on a small peptide substrate has been developed. A number of putative small fluorogenic peptide substrates were prepared and evaluated and optimized compounds showed reasonable rates of cleavage but required high enzyme concentrations which limited their value. A modified peptide substrate incorporating a less sterically demanding self-immolative element was designed and synthesized and was shown to have enhanced properties useful for evaluating inhibitors of ATG4B. Substrate cleavage was readily monitored and was linear for up to 4h but enzyme concentrations of about ten-fold higher were required compared to assays using protein substrate LC3 or analogs thereof (such as FRET-LC3). Several known inhibitors of ATG4B were evaluated using the small peptide substrate and gave IC50 values 3-7 fold higher than previously obtained values using the FRET-LC3 substrate.

Construction and characterization of kilobasepair densely labeled peptide-DNA.

Directed assembly of biocompatible materials benefits from modular building blocks in which structural organization is independent of introduced functional modifications. For soft materials, such modifications have been limited. Here, long DNA is successfully functionalized with dense decoration by peptides. Following introduction of alkyne-modified nucleotides into kilobasepair DNA, measurements of persistence length show that DNA mechanics are unaltered by the dense incorporation of alkynes (∼1 alkyne/2 bp) and after click-chemistry attachment of a tunable density of peptides. Proteolytic cleavage of densely tethered peptides (∼1 peptide/3 bp) demonstrates addressability of the functional groups, showing that this accessible approach to creating hybrid structures can maintain orthogonality between backbone mechanics and overlaid function. The synthesis and characterization of these hybrid constructs establishes the groundwork for their implementation in future applications, such as building blocks in modular approaches to a range of problems in synthetic biology.

A novel class of broad-spectrum active-site-directed 3C-like protease inhibitors with nanomolar antiviral activity against highly immune-evasive SARS-CoV-2 Omicron subvariants.

Antivirals with broad coronavirus activity are important for treating high-risk individuals exposed to the constantly evolving SARS-CoV-2 variants of concern (VOCs) as well as emerging drug-resistant variants. We developed and characterized a novel class of active-site-directed 3-chymotrypsin-like protease (3CLpro) inhibitors (C2-C5a). Our lead direct-acting antiviral (DAA), C5a, is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro. The compounds C2-C5a exhibit broad-spectrum activity against Omicron subvariants (BA.5, BQ.1.1, and XBB.1.5) and seasonal human coronavirus-229E infection in human cells. Notably, C5a has median effective concentrations of 30-50 nM against BQ.1.1 and XBB.1.5 in two different human cell lines. X-ray crystallography has confirmed the unique binding modes of C2-C5a to the 3CLpro, which can limit virus cross-resistance to emerging Paxlovid-resistant variants. We tested the effect of C5a with two of our newly discovered host-directed antivirals (HDAs): N-0385, a TMPRSS2 inhibitor, and bafilomycin D (BafD), a human vacuolar H+-ATPase [V-ATPase] inhibitor. We demonstrated a synergistic action of C5a in combination with N-0385 and BafD against Omicron BA.5 infection in human Calu-3 lung cells. Our findings underscore that a SARS-CoV-2 multi-targeted treatment for circulating Omicron subvariants based on DAAs (C5a) and HDAs (N-0385 or BafD) can lead to therapeutic benefits by enhancing treatment efficacy. Furthermore, the high-resolution structures of SARS-CoV-2 3CLpro in complex with C2-C5a will facilitate future rational optimization of our novel broad-spectrum active-site-directed 3C-like protease inhibitors.

Recent progress in biologically active indole hybrids: a mini review.

The indole moiety is one of the most widespread heterocycles found in both natural products and biological systems. Indoles have important biological activities including anticancer, antioxidant, anti-inflammatory, antifungal, anticholinesterase, and antibacterial properties. Scientists are therefore interested in the synthesis of biologically active indole-based hybrids such as indole-coumarin, indole-chalcone, indole-isatin, indole-pyrimidine and so on, with the aim of improving activity, selectivity, and mitigating side effects. This review will discuss the newly synthesized indole-based hybrids along with their biological activity which will be useful in drug discovery and development.

Design and construction of the lawnmower, an artificial burnt-bridges motor.

Molecular motors of the cell are protein-based, nanoscale machines, which use a variety of strategies to transduce chemical energy into mechanical work in the presence of a large thermal background. The design and construction of artificial molecular motors is one approach to better understand their basic physical principles. Here, we propose the concept of a protein-based, burnt-bridges ratchet, inspired by biological examples. Our concept, the lawnmower, utilizes protease blades to cleave peptide substrates, and uses the asymmetric substrate-product interface arising from productive cleavage to bias subsequent diffusion on the track (lawn). Following experimental screening to select a protease to act as the motor's blades, we chemically couple trypsin to quantum dots and demonstrate activity of the resulting lawnmower construct in solution. Accompanying Brownian dynamics simulations illustrate the importance for processivity of correct protease density on the quantum dot and spacing of substrates on the track. These results lay the groundwork for future tests of the protein-based lawnmower's motor performance characteristics.

Development of fluorescent substrates and assays for the key autophagy-related cysteine protease enzyme, ATG4B.

The cysteine protease ATG4B plays a role in key steps of the autophagy process and is of interest as a potential therapeutic target. At an early step, ATG4B cleaves proLC3 isoforms to form LC3-I for subsequent lipidation to form LC3-II and autophagosome membrane insertion. ATG4B also cleaves phosphatidylethanolamine (PE) from LC3-II to regenerate LC3-I, enabling its recycling for further membrane biogenesis. Here, we report several novel assays for monitoring the enzymatic activity of ATG4B. An assay based on mass spectrometric analysis and quantification of cleavage of the substrate protein LC3-B was developed and, while useful for mechanistic studies, was not suitable for high throughput screening (HTS). A doubly fluorescent fluorescence resonance energy transfer (FRET) ligand YFP-LC3B-EmGFP (FRET-LC3) was constructed and shown to be an excellent substrate for ATG4B with rates of cleavage similar to that for LC3B itself. A HTS assay to identify candidate inhibitors of ATG4B utilizing FRET-LC3 as a substrate was developed and validated with a satisfactory Z' factor and high signal-to-noise ratio suitable for screening small molecule libraries. Pilot screens of the 1,280-member library of pharmacologically active compounds (LOPAC(™)) and a 3,481-member library of known drugs (KD2) gave hit rates of 0.6% and 0.5% respectively, and subsequent titrations confirmed ATG4B inhibitory activity for three compounds, both in the FRET and mass spectrometry assays. The FRET- and mass spectrometry-based assays we have developed will allow for both HTS for inhibitors of ATG4B and mechanistic approaches to study inhibition of a major component of the autophagy pathway.