Biology subjects

SARKAR, S. K.

Publications and source records attributed to SARKAR, S. K..

4 recordsLinked to original sources

bioRxiv2026

Drug design using unique conformations to preferentially target a specific site on collagen-bound MMP1

Precise site-specific drug design remains a challenge in structure-based drug discovery. Most existing approaches screen for ligands to target binding pockets on a protein surface based on static structures obtained from techniques such as X-ray, NMR, cryo-EM, and AlphaFold. However, the structure-function paradigm is, in reality, a structure-dynamics-function relationship that determines a proteins binding and activity. As such, drug screening or design without evaluating binding competition across the protein surface or considering the receptors dynamic, substrate-dependent conformational states is incomplete. Substrate-specific unique protein conformations are underexplored and offer novel opportunities for selective therapeutic targeting, though systematic workflows for identifying and exploiting such sites remain limited. Previously, we showed that collagen alters matrix metalloprotease-1 (MMP1) dynamics and that R405 is an allosteric residue on the MMP1 surface that exhibits strong dynamic correlations with its active site. Here, we present a substrate-specific allosteric drug-design framework that targets specific sites on a protein, using collagen-bound MMP1 as a model system. We determined the conformational dynamics of free and collagen-bound MMP1 using all-atom molecular dynamics (MD) simulations and categorized conformations into clusters of similar conformations. We then compared and identified unique conformations that occur only in collagen-bound MMP1 to design drugs against them using a machine-learning approach. The top three unique clusters were used to generate approximately 150,000 candidate compounds that were then screened against both the R405-centered region and all detectable binding pockets across the MMP1 surface. We have found several compounds that bind preferentially around R405 by at least 0.3 kcal/mol relative to competing sites across the surface. This strategy establishes a generalizable framework for designing ligands that preferentially target substrate-specific allosteric sites, providing new opportunities for precision therapeutics that modulate proteins in their biologically relevant functional states. Simple SummaryIn this paper, we establish a substrate-specific allosteric drug-design strategy that integrates all-atom molecular dynamics simulations, conformational clustering, machine-learning-based ligand design, and surface-wide binding-selectivity screening, using collagen-bound MMP1 as a model system. We show that collagen binding reshapes the conformational ensemble of MMP1, creating unique conformational states that are absent or inaccessible in the free enzyme. By identifying these substrate-specific conformations, generating ligands based on the corresponding dynamic fingerprints around the collagen-specific allosteric residue R405, and screening compounds across all binding pockets on the MMP1 surface, we demonstrate preferential targeting of the collagen-specific site relative to competing pockets. These results establish a generalizable framework for designing ligands that selectively recognize biologically relevant substrate-bound conformations rather than static protein structures alone. Substrate-specific allosteric targeting may enable selective modulation of individual protein functions while minimizing off-target interactions, providing new opportunities for precision therapeutics against dynamic protein systems.

biophysics↗

bioRxiv2025

Transcriptomic landscape of Gallbladder cancer reveals altered pathways related to cell cycle and Aurora kinase

BackgroundGallbladder cancer (GBC) is a rare but aggressive biliary tract malignancy. This study explores the transcriptomic profile of GBC to identify differentially expressed genes (DEGs) and dysregulated pathways involved in its pathogenesis. MethodsRNA sequencing was performed on 13 GBC tumors and 6 matched controls. Functional enrichment analysis as well as WGCNA were used to identify dysregulated pathways, functionally relevant gene modules and hub genes. Key targets were validated in patient tissues and cell lines. ResultsA total of 621 DEGs were identified (247 upregulated, 374 downregulated). Gene set enrichment analysis revealed activation of E2F targets and G2/M checkpoint, with downregulation of bile acid metabolism and estrogen response pathways. A tumor grade-correlated WGCNA module was enriched in cell cycle genes. TPX2 emerged as a central hub gene. Inhibitors of aurora kinase, TPX2 dependent enzyme, significantly reduced proliferation, migration, and invasion in GBC cells. High-grade tumors confirmed elevated Aurora kinase expression. ConclusionsThis first transcriptomic analysis of GBC in South-East Asian Indians uncovers key drivers like TPX2 and Aurora kinases in disease progression. The study highlights cell cycle dysregulation and sex-linked signatures, offering insights for biomarker discovery and targeted therapies.

cancer biology↗

bioRxiv2025

The substrate changes matrix metalloprotease-1 dynamics, allostery, and ligand binding affinity

Using single-molecule experiments, ensemble assays, and Molecular Dynamics (MD) simulations, we showed that collagen actively influences MMP1 interdomain dynamics. This paper extends the previous experimental work to validate MD simulations and gain more insights into MMP1-collagen interactions. We include virtual screening with free energy calculations to uncover key insights regarding substrate-dependent dynamics, allostery, and ligand binding. Our findings reveal that MMP1 interdomain dynamics are influenced by the strain in collagen, and collagen-specific allosteric residues exhibit strong correlations with the active site. Additionally, allosteric paths connecting these residues to the active site bypass the linker region. Collagen also induces changes in the configurations of the catalytic motif. Furthermore, MD simulation and virtual screening identify molecules that bind to allosteric residues, alter MMP1 dynamics, and potentially change activity. Free energy calculations highlight the essential role of the substrate in small molecule screening against a protein. We have developed a comprehensive framework for identifying substrate-specific allosteric residues in MMP1 and screening small molecules incorporating protein dynamics in the presence of substrates. These results significantly impact our understanding of MMP1 interactions with collagen fibrils and pave the way for selectively modulating specific MMP1 functions without altering its other functions in future applications. SignificanceIn this paper, we validated all-atom simulations of free and collagen-bound MMP1 and gained insights into MMP1 function. We have found that collagen changes dynamics, allostery, and ligand binding in MMP1. Collagen strain, known to be age-dependent, also changes MMP1 dynamics and ligand binding. Interdomain allosteric paths do not go through the linker connecting the MMP1 domains. Instead, paths from the collagen-specific allosteric residue to the active site have non-bonded interactions. Screening of small molecules against collagen-specific MMP1 residue suggests that collagen alters ligand binding to MMP1. These results will significantly impact understanding MMPs and small molecule screening against proteins. Substrate-specific allosteric residues may enable controlling one function of MMPs without altering their other functions, leading to fewer side effects.

biophysics↗

bioRxiv2021

Quantification of allosteric communications in matrix metalloprotease-1 on alpha-synuclein aggregates and substrate-dependent virtual screening

Alpha-synuclein (aSyn) has implications in pathological protein aggregations observed in neurodegenerative disorders, including Parkinsons and Alzheimers diseases. There are currently no approved prevention and cure for these diseases. In this context, matrix metalloproteases (MMPs) provide an opportunity because MMPs are broad-spectrum proteases and cleave aSyn. Previously, we showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, MMP1 activity, and ligands. However, allosteric communications in MMP1 on aSyn-induced aggregates have not been explored. Here we report quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations, with the two domains well-separated. These open conformations are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics. The best-fit parameters for a Gaussian fit to the distributions of FRET values provide the two states. The ratio of the kinetic rates between the two states comes from the ratio of fitted areas around the two states. The decay rate of an exponential fit to the correlations between FRET values provides the sum of the kinetic rates. Since a crystal structure of aSyn-bound MMP1 is not available, we performed molecular docking of MMP1 with aSyn using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to determine the most appropriate pose. We performed virtual screening against the potential ligand-binding sites on the appropriate aSyn-MMP1 binding pose and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading pathological aggregates in neurodegeneration by targeting MMPs. SignificanceWe have quantified MMP1 interdomain dynamics on aSyn-induced aggregates by a two-state Poisson process. Histograms and correlations of FRET values determine the kinetic rates of interconversion between the two states. We quantify the conformational dynamics of the whole MMP1 and allosteric communications by the two-dimensional matrix of correlations between every pair of amino acids from experimentally-validated all-atom simulations. The two-dimensional correlations lead to a Gray Level Co-occurrence Matrix and a measure of Shannon entropy describing the conformational fluctuations. As such, we address the quantification of allosteric communications, a leading challenge in defining allostery. We report that the potential ligand-binding sites and lead molecules change for MMP1 upon binding alpha-synuclein and depend on the binding pose selected. This suggests that one needs to take the substrate into account while targeting MMPs.

biophysics↗