Biology subjects

Munshi, S.

Publications and source records attributed to Munshi, S..

8 recordsLinked to original sources

bioRxiv2025

Potent broad-spectrum anti-coronaviral frameshift inhibitors from virtual screen of RNA binding

Coronavirus genomes contain an RNA pseudoknot that directs -1 programmed ribosomal frame-shifting (-1 PRF) to control expression of viral proteins crucial for replication. Ligands that inhibit -1 PRF can thus attenuate viral propagation and have potential as drugs for limiting corona-virus infections. To search for novel small-molecule frameshift inhibitors with anticoronaviral activity, we computationally screened over 14 million compounds for binding to the SARS-CoV-2 pseudoknot, followed by experimental validation of the top hits for inhibition of -1 PRF and viral replication. We identified multiple potent -1 PRF inhibitors, effective at nM concentrations, some of which significantly suppressed SARS-CoV-2 replication in cell culture. Several compounds also inhibited -1 PRF in multiple representative bat coronaviruses, indicating broad-spectrum activity. These results showcase the promise of viral RNA structures like frameshift-stimulatory pseudoknots as targets for broad-spectrum antiviral drugs.

biophysics↗

bioRxiv2023

Computationally Driven Discovery and Characterization of SIRT3 Activating Compounds that Fully Recover Catalytic Activity under NAD+ Depletion

Mammalian sirtuins (SIRT1-SIRT7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases that play critical roles in lifespan and age-related diseases. The physiological importance of sirtuins has stimulated intense interest in designing sirtuin activating compounds. However, except for allosteric activators of SIRT1-catalyzed reactions that are limited to specific substrates, methodologies for the rational design of sirtuin activating compounds -- including compounds that activate mitochondrial sirtuins implicated in the age-related decline of cellular metabolism -- have been lacking. Here, we use computational high-throughput screening methodologies and a biophysical model for activation of the major mitochondrial sirtuin SIRT3 to identify novel small molecule activators of the human SIRT3 enzyme from a 1.2 million compound library. Unlike previously reported SIRT3 activators like Honokiol, which only transiently upregulate SIRT3 under non-steady state conditions and reduce the steady state catalytic efficiency of the enzyme, several of the novel compounds identified here are potent SIRT3 activators in both the steady and non-steady states. Two such compounds can almost double the catalytic efficiency of the enzyme with respect to NAD+, which would be sufficient to almost entirely compensate for the loss in SIRT3 activity that occurs due to the reduction in mitochondrial coenzyme concentration associated with aging, and display AC50s (concentrations of half-maximal activation) as low as 100 nM. The current work thus reports first-in-class, non-allosteric steady state activators that activate SIRT3 through a novel, mechanism-based mode of activation and that may be developed further for therapeutic applications.

biochemistry↗

bioRxiv2022

Functional regulation of an intrinsically disordered protein via a conformationally excited state

A longstanding goal in the field of intrinsically disordered proteins (IDP) is to characterize their structural heterogeneity and pinpoint the role of this heterogeneity in IDP function. Here, we use multinuclear chemical exchange saturation (CEST) NMR to determine the structure of a thermally accessible globally folded excited state in equilibrium with the intrinsically disordered native ensemble of a bacterial transcriptional regulator CytR. We further provide evidence from double resonance CEST experiments that the excited state, which structurally resembles the DNA-bound form of CytR, recognizes DNA by means of a folding-before-binding conformational selection pathway. The disorder-to-order regulatory switch in DNA recognition by natively disordered CytR therefore operates through a dynamical variant of the lock-and-key mechanism where the structurally complementary conformation is transiently accessed via thermal fluctuations. One-Sentence SummaryThe intrinsically disordered cytidine repressor binds DNA via a folding-before-binding conformational selection mechanism

biophysics↗

bioRxiv2021

Small-molecule ligands can inhibit -1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases since 2000 have high-lighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored a group of 6 small-molecule ligands, evaluating their activity against the frameshift signals from a panel of representative bat CoVs--the most likely source of future zoonoses--as well as SARS-CoV-2 and MERS-CoV. We found that whereas some ligands had notable activity against only a few of the frameshift signals, the serine protease inhibitor nafamostat suppressed -1 PRF significantly in several of them, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing the frameshift signal as a viable target for developing pan-coronaviral therapeutics.

biochemistry↗

bioRxiv2020

Structural dynamics of the SARS-CoV-2 frameshift-stimulatory pseudoknot reveal topologically distinct conformers

The RNA pseudoknot that stimulates -1 programmed ribosomal frameshifting in SARS coronavirus-2 (SARS-CoV-2) is a possible drug target. To understand how this 3-stemmed pseudoknot responds to the mechanical tension applied by ribosomes during translation, which is thought to play a key role during frame-shifting, we probed its structural dynamics under tension using optical tweezers. Unfolding curves revealed that the frameshift signal formed multiple different structures: at least two distinct pseudoknotted conformers with different unfolding forces and energy barriers, as well as alternative stem-loop structures. Refolding curves showed that stem 1 formed first in the pseudoknotted conformers, followed by stem 3 and then stem 2. By extending the handle holding the RNA to occlude the 5' end of stem 1, the proportion of the different pseudoknot conformers could be altered systematically, consistent with structures observed in cryo-EM images and computational simulations that had distinct topologies: the 5' end of the RNA threaded through the 3-helix junction to form a ring-knot, or unthreaded as in more standard H-type pseudoknots. These results resolve the folding mechanism of the frameshift signal in SARS-CoV-2 and highlight the dynamic conformational heterogeneity of this RNA, with important implications for structure-based drug-discovery efforts.

biophysics↗

bioRxiv2020

New mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy.

Fn1 fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live imaging and single-molecule localization microscopy (SMLM) are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing 6-11 Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, wherein nanodomains are spaced at the average periodicity of 105{+/-}17 nm. Periodical Fn1 nanodomain arrays are bona fide fibrils: they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. FUD, a bacterial peptide that binds Fn1 N-terminus and disrupts Fn1 fibrillogenesis does not disrupt the formation of Fn1 nanodomains, instead, it blocks the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis.

cell biology↗

bioRxiv2020

Anti-frameshifting ligand active against SARS coronavirus-2 is resistant to natural mutations of the frameshift-stimulatory pseudoknot

The coronavirus SARS-CoV-2 causing the COVID-19 pandemic uses −1 programmed ribosomal frameshifting (−1 PRF) to control the expression levels of key viral proteins. Because modulating −1 PRF can attenuate viral propagation, ligands binding to the viral RNA pseudoknot that stimulates −1 PRF may prove useful as therapeutics. Mutations in the pseudoknot have been observed over the course of the pandemic, but how they affect −1 PRF and the activity of inhibitors is unknown. Cataloguing natural mutations in all parts of the SARS-CoV-2 pseudoknot, we studied a panel of 6 mutations in key structural regions. Most mutations left the −1 PRF efficiency unchanged, even when base-pairing was disrupted, but one led to a remarkable three-fold decrease, suggesting that SARS-CoV-2 propagation may be less sensitive to modulation of −1 PRF efficiency than some other viruses. Examining the effects of one of the few small-molecule ligands known to suppress −1 PRF significantly in SARS-CoV, we found that it did so by similar amounts in all SARS-CoV-2 mutants tested, regardless of the basal −1 PRF efficiency, indicating that the activity of anti-frameshifting ligands can be resistant to natural pseudoknot mutations. These results have important implications for therapeutic strategies targeting SARS-CoV-2 through modulation of −1 PRF.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Nonallosteric Sirtuin Enzyme Activation: Characterization of Hit Compounds

Mammalian sirtuins (SIRT1-SIRT7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases that play critical roles in lifespan and age-related diseases. The physiological importance of sirtuins has stimulated intense interest in designing sirtuin-activating compounds. However, except for allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates, a general framework for the design of sirtuin-activating compounds has been lacking. Recently, we introduced a general mode of sirtuin activation that is distinct from the known modes of enzyme activation, establishing biophysical properties of small molecule modulators that can, in principle, result in enzyme activation for various sirtuins and substrates. Here, we characterize small molecules reported in the literature to activate the SIRT3 enzyme using a variety of computational, biochemical and biophysical techniques including protein-ligand docking, molecular dynamics simulation, nonlinear reaction dynamics simulation, kinetic assays and thermodynamic assays with multiple substrates and protocols. In particular, we identify the mechanism of action of the compound honokiol on the human SIRT3 enzyme, modeling its effect on active site conformational degrees of freedom and demonstrating how it nonallosterically activates the human SIRT3 enzyme under physiologically relevant conditions. We show that honokiol constitutes a hit compound for the design of a new generation of nonallosteric activators that can activate SIRT3 through the proposed mechanism-based mode of activation.

biophysics↗