Biology subjects

Chatterjee, D.

Publications and source records attributed to Chatterjee, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Glial Mutant Huntingtin Expression Initiates a Progressive Pathological Cascade Toward Neuronal Involvement in Drosophila

The mechanisms of involvement of glial cells in the progression of Huntingtons disease remain sparingly elucidated. Using the Drosophila melanogaster model, we investigated the temporal course of the disease pathogenesis induced by pan-glial expression of HTTex1pQ93 in comparison to the temporal course of pathology induced by pan-neuronal expression. Our behavioural data were supported by quantitative confocal microscopy, quantification of aggregate formation and its morphometry, Manders M2 colocalization index, and correlative holotomography. Expression of mHTT in glial cells led to developmental defects and subsequent delayed onset of motor dysfunction and death with increasing amounts of HTT and changes in the immunoreactivity of glial cells. In spite of the progressive increase in HTT accumulation, glial aggregates showed stability after their initial formation, while neuronal mHTT revealed distinct age-dependent remodelling of its aggregates. Importantly, there was an initial preference of glial mHTT for glial compartments followed by a progressively increasing number of HTT in the neurons with aging. Correlative holotomography provided spatial evidence of the neuronal association of HTT. Together, these findings demonstrate that glial mHTT establishes a distinct progressive pathological trajectory characterised by evolving glial pathology, increasing neuronal involvement, and delayed functional decline, highlighting glia as an active component of HD-like disease progression.

neuroscience↗

bioRxiv2026

REV-ERBα/β Targeting Transcriptionally Reprograms HIV-Infected CD4+ T-Cells for Increased Viral Reactivation but Limited Virion Spread

The circadian clock repressors REV-ERB/{beta} control rhythmic gene expression and inhibit HIV-1 transcription. Whether REV-ERB/{beta} modulate the HIV-1 replication cycle beyond transcription remains unknown. Here, we demonstrate that memory CD4+ T-cells predominantly express the REV-ERB{beta} isoform ex vivo and that T-cell receptor (TCR) triggering downregulates both REV-ERB/{beta} expression, with levels of REV-ERB/{beta} mRNA being lower in ART-treated people with HIV (PWH) receiving antiretroviral therapy (ART) compared to people without HIV (PWoH) before/after TCR triggering. In single-round infection, the REV-ERB/{beta} antagonist SR8278 facilitated HIV-1 reverse transcription, integration, and intracellular HIV-p24 expression, but limited virion release. Moreover, SR8278 downregulated CCR5 mRNA expression, inhibited R5-tropic HIV-1 replication in vitro, and limited viral outgrowth in CD4+ T-cells from ART-treated PWH. Finally, genome-wide RNA-sequencing and functional validations revealed HIV-1 restriction/dependency factors that represent novel putative REV-ERB/{beta} targets. Thus, pharmacological inhibition of REV-ERB/{beta} uniquely combines a latency reversal activity with the inhibition of progeny virion spread.

immunology↗

bioRxiv2026

Structural basis for the ATP-dependence of omegasome biogenesis by DFCP1

Autophagosome formation begins at phosphatidylinositol 3-phosphate-enriched endoplasmic reticulum (ER) subdomains termed omegasomes. DFCP1/ZFYVE1 is recruited to omegasomes through its FYVE domains and has recently been shown to function as an ATPase involved in omegasome constriction and autophagosome biogenesis. However, the structural basis of DFCP1 ATPase activity and how nucleotide-dependent conformational states regulate omegasome dynamics remain unknown. Here, we determined the crystal structures of the DFCP1 ATPase domain in complex with either ADP or the non-hydrolyzable ATP analogue, AppNHp at near atomic resolution. We employed structure-guided mutagenesis to define residues required for DFCP1 function in cells. The structures reveal that the active site contains a trans-acting Arg271 finger coordinating the {psi}-phosphate and trans-acting His323 that stacks on the ATP adenine ring. ATP hydrolysis results in a conformational switch in the vicinity of Arg271, while contacts with His323 persist in the presence of ADP. Biochemical analyses of DFCP1 and its mutants show that DFCP1 forms ATP-dependent microdomains on membranes. Disruption of His323 and Arg271 uncouples nucleotide binding from ATP hydrolysis and alters the oligomeric state of the protein. Live-cell imaging of DFCP1 knockout cells reconstituted with wild-type or mutant DFCP1 further demonstrates that these biochemical defects translate into distinct omegasome phenotypes. Together, our data provides a structural-function framework for DFCP1 ATPase activity and reveals how distinct catalytic elements control omegasome dynamics in vivo. We propose that nucleotide-dependent assembly and hydrolysis-driven conformational changes enable DFCP1 to regulate omegasome formation and its progression toward autophagosome closure. Significance StatementDFCP1 organizes the ER subdomains known as omegasomes, which are sites of autophagosome biogenesis. DFCP1 is an ATPase whose catalytic activity is required for function, but the precise role of its ATPase activity is unknown. Crystal structures of the DFCP1 ATPase domain in the presence of a non-hydrolyzable ATP analogue and ADP reveal an Arg finger that operates in trans, such that dimerization is required for ATP hydrolysis, and ATP hydrolysis destabilizes dimers. Dimerization is also promoted by an adenine base-stacking interaction in trans with H323. These residues are important for DFCP1 clustering on membranes and omegasome constriction, clarifying that the role of ATP is to promote the dimerization of the ATPase domain and thereby control the organization of DFCP1 on membranes.

biochemistry↗

bioRxiv2026

Rv0783c of Mycobacterium tuberculosis acts as a proton-motive force dependent multidrug efflux transporter involved in the efflux of structurally unrelated antibiotics and enhancing biofilm formation

Despite multiple treatment strategies and extensive research on resistance mechanisms, tuberculosis (TB) remains a major global health threat, largely because of the rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. Among various mechanisms complicating the situation, active antibiotic export via efflux pumps is particularly significant, yet largely unexplored. Mycobacterium sp. encodes numerous transporters, many of which are overexpressed in clinical isolates or under drug stress. Here, we examined the possible role of Rv0783c, a putative transporter that is reportedly overexpressed in drug-stressed conditions. Rv0783c conferred resistance to multiple structurally diverse antibiotics, fluoroquinolones and anti-TB drugs in the heterologous hosts, namely, Escherichia coli and Mycobacterium smegmatis. Reduced drug accumulation and active efflux of ethidium bromide (EtBr) confirmed its transport activity, which in turn gets nullified upon using the proton-motive force blocker, CCCP. On the other hand, its expression enhanced biofilm formation, linking antibiotic resistance to persistence-associated phenotype. Furthermore, site-directed mutagenesis confirmed the presence of crucial interacting residues with antibiotics that were identified by in silico analysis. Overall, we demonstrate the role of Rv0783c in the extrusion of first and second-line anti-TB drugs and enhancing biofilm formation.

microbiology↗

bioRxiv2026

Probing the role of residues lining the active site in the generation of glucose-tolerant variants of a fungal GH1 enzyme

The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a {beta}-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was [~]0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance ([~]1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails.

biochemistry↗

bioRxiv2025

Substitution of a valine to glutamic acid in the omega-like loop of MSMEG_6194 of Mycobacterium smegmatis interchanges its activity from DD-carboxypeptidase to beta-lactamase

The genome of Mycobacterium smegmatis encodes numerous penicillin-interacting enzymes, and analysing their functions provides insights into the evolutionary mechanisms behind beta-lactam resistance in mycobacteria. In this study, we characterised one such enzyme, MSMEG_6194, annotated as a putative beta-lactamase. Although MSMEG_6194 shares structural similarity with class A beta-lactamases, it showed no detectable beta-lactamase activity under the tested conditions. Heterologous expression of MSMEG_6194 in Escherichia coli and{Delta} msmeg_6194 deleted strains of M. smegmatis did not confer significant resistance to beta-lactams, and the purified protein failed to hydrolyse nitrocefin either. However, ectopic expression of MSMEG_6194 partly restores the morphological defects in seven PBP-deleted E. coli strains, and the purified enzyme successfully cleaves the terminal D-alanine from a pentapeptide substrate, confirming its DD-carboxypeptidase activity. Structural analysis revealed the absence of a conserved glutamic acid residue in the omega-loop, which is critical for beta-lactamase catalysis in class A beta-lactamase. Substituting this residue (V139E mutant) imparts beta-lactamase activity though significantly reduces DD-carboxypeptidase function. Overall, these findings establish MSMEG_6194 as a DD-carboxypeptidase and demonstrate how a single amino acid change can alter catalytic preference, shedding light on the evolutionary transition from DD-Carboxypeptidases to beta-lactamases in mycobacteria.

molecular biology↗

bioRxiv2025

Candida albicans infiltrates colon and rectal cancers causing therapeutic resistance and decreased survival

The microbiome is increasingly recognized as a modifier of cancer progression and therapy response, yet the role of intratumoral fungi remains poorly defined. Here, we identify Candida albicans colonization within human colorectal tumors as a predictor of reduced survival and impaired radiation response. Leveraging the Oncology Research Information Exchange Network (ORIEN) cohort, we show that high intratumoral Candida burden is associated with decreased survival across multiple gastrointestinal cancers, with the strongest treatment-specific effect in rectal cancer patients receiving radiotherapy. This observation was validated in independent rectal cancer cohorts using RNA sequencing and quantitative PCR. In immune-competent murine colorectal cancer models, oral gavage of C. albicans resulted in intratumoral colonization, accelerated tumor growth, and radiation resistance, effects not observed with Saccharomyces cerevisiae or PBS controls. Colonized tumors exhibited increased hypoxia, altered metabolic and transcriptional programs, and distinct expression of genes linked to cytokine signaling and cell survival. Hypoxia conditioned C. albicans secreted metabolites that directly conferred radiation resistance to colorectal cancer cells in vitro, implicating a cancer cell intrinsic mechanism independent of immune signaling. Untargeted metabolomics revealed enrichment of nucleosides and lipid oxidation intermediates under hypoxia, suggesting that C. albicans metabolites may provide substrates facilitating tumor recovery after irradiation. These findings establish C. albicans as a causal modifier of tumor biology and radiation response, highlighting intratumoral fungi as future potential therapeutic targets. Modulating fungal colonization or metabolism may improve radiotherapy outcomes and broaden our understanding of interactions between microbes and tumors.

cancer biology↗

bioRxiv2025

Putative MFS transporter Rv1250 of Mycobacterium tuberculosis is involved in multidrug efflux activity

Drug-resistant Mycobacterium tuberculosis is one of the leading causes of global mortality. Mechanisms, such as slow uptake of drugs along with cell wall impermeability and active efflux, are some of the concerning reasons leading to drug resistance. Efflux pumps actively transport a wide variety of drugs and toxins away from the target site, which is considered an emerging cause for the failure of anti-tubercular medications and treatment. In this study, we report that the ability of Rv1250, a probable MFS-type transporter, influences the extrusion of multiple structurally unrelated classes of drugs, enhances the biofilm formation in E. coli and Mycobacterium smegmatis, and facilitates the survival of M. smegmatis cells inside the macrophage during antibiotic stress. Interestingly, in trans, the expression of rv1250 decreased the susceptibility of host cells to several structurally unrelated antibiotics, ranging from fluoroquinolones to aminoglycosides, beta-lactams, and anti-tubercular drugs, thus indicating its involvement in imparting intrinsic drug tolerance. In addition, the increased efflux of EtBr, norfloxacin, and Bocillin FL from host cells expressing rv1250 was revealed by the hosts ability to confer a lower level of antibiotic accumulation. Moreover, the expression of rv1250 resulted in the enhancement of biofilm formation. Overall, we conclude that Rv1250 of Mycobacterium tuberculosis might facilitate the survival of host cells under antimicrobial stress. One sentence summaryRole of Rv1250 as an efflux pump

microbiology↗

bioRxiv2025

Reprogrammed E. coli for secretion of thermophilic cellulase cocktail for seawater-compatible lignocellulosic bioprocessing

The development of efficient cellulase systems is crucial for sustainable lignocellulosic biorefining. In this study, we engineered Escherichia coli to co-express and secrete a thermophilic cellulase cocktail consisting of a Bacillus licheniformis processive endoglucanase (H1AD14), a Chaetomium thermophilum cellobiohydrolase (G0SAE6), and a glucose-tolerant engineered {beta}-glucosidase B8CYA8tm (V169C/E173L/I246A B8CYA8) from Halothermothrix orenii. Fusion with the AnsB secretion tag enabled extracellular yields of 0.2-0.4 g/L for each enzyme, bypassing costly purification while retaining activity. The secretion was confirmed via immunoblotting using anti-His antibody, and catalytic activity was validated through enzymatic assays. The absence of E. coli native periplasmic maltose-binding protein (detected with anti-MBP antibody) in the extracellular medium ruled out cell lysis, confirming protein secretion. Using the Golden Gate assembly, we constructed and systematically evaluated synergistic enzyme combinations for hydrolytic performance. By optimizing secretion conditions, including media, inducer concentration, and incubation temperature, the total yield of secreted enzyme cocktail increased from 0.31 g/L to 0.74 g/L. The secreted cocktail effectively hydrolysed diverse lignocellulosic substrates and exhibited 100 % activity in seawater, demonstrating potential for freshwater-independent biorefining. The work addresses key economic and environmental challenges in biomass conversion by integrating secretion engineering, thermostability, and seawater tolerance into a single scalable system.

bioengineering↗

bioRxiv2025

Photothrombotic Ischemic Thalamic Stroke in Mice Recapitulates Spontaneous Pain Features of Central Post-Stroke Pain in Humans

Central post-stroke pain (CPSP) is a highly distressing condition that develops in 50% of people who suffer a thalamic stroke, and is typically unresponsive to current clinical treatments. Hypoxic damage to the ventral posterolateral (VPL) and ventral posteromedial (VPM) sensory thalamic nuclei, in particular, precipitates CPSP. One barrier to developing treatments for CPSP is the lack of preclinical models of thalamic ischemic stroke. In this study, we present a novel mouse model of CPSP induced through targeted photothrombotic ischemia. After eliciting hypoxia in the sensory thalamus of male mice, we assessed pain behaviors over a four-week period. Stroke-affected mice exhibited a persistent spontaneous facial grimace from day four to week four post-stroke, indicative of pain. Hind-paw mechanical hypersensitivity indicative of altered nociception, characteristic of VPL and VPM hemorrhagic CPSP models, was not detected in our model. Immunofluorescence analysis revealed increased activated microglia (Iba1) and reactive astrocytes (GFAP). Iba1 fluorescence intensity in the VPL thalamus-but not the VPM thalamus-correlated with the severity of facial grimace at four weeks post-stroke. Clustering based on behavioral phenotypes identified a subpopulation of mice in which grimace pain spontaneously resolved, by four weeks post-stroke, relative to sham controls, suggesting that this model can be used to understand how stroke recovery may influence pain chronification. This model provides a valuable tool to investigate the cellular and circuit mechanisms underlying CPSP after an ischemic thalamic stroke. Significance StatementResearch into central post-stroke pain (CPSP) is hindered by the lack of preclinical models that match the clinical presentation of the condition. Although 87% of clinical strokes are ischemic, current CPSP preclinical models induce hemorrhagic strokes. To our knowledge, our mouse model is the first to induce CPSP by photothrombosis in the ventral posterolateral and posteromedial thalamus. Moreover, our study quantifies spontaneous pain, using a facial grimace score, in addition to nociception, whereas previous CPSP studies have relied exclusively on nociceptive tests. Finally, our model recapitulates some of the variability observed in clinical CPSP. We anticipate that this model will help dissect the roots of chronic pain after a thalamic stroke, and facilitate development of novel therapies for CPSP.

neuroscience↗

bioRxiv2025

Selectivity profiles and substrate recognition of Rab phosphorylating kinases

The Rab GTPase switch-2 region is a hotspot for post-translational modifications. Its phosphorylation can determine whether individuals develop Parkinsons disease or not. Other modifications of the same region are catalysed by enzymes from bacterial pathogens when they infect human cells. Here, we profiled a set of kinases including LRRK1, LRRK2, DYRK1A, MST1, and TBK1 for their capability of phosphorylating Rab GTPases. We identified several novel kinase:Rab pairs, such as LRRK1:Rab43 and TBK1:Rab29. Further, we comprehensively assessed what makes a Rab GTPase a good kinase substrate, considering the Rab nucleotide binding state and the Rab primary sequence. In a systematic mutational study, Rab variants with modulated phosphorylation properties were established, leading to the identification of a LRRK2 recognition patch in the Rab 3 helix. A Glu to Arg exchange in that patch increased the phosphorylation 18-fold indicating that Rabs are suboptimal LRRK2 substrates. Given that this effect is also observed in a cellular model, we propose that our variants will be excellent tools for analysing the physiological function of Rab phosphorylation.

biochemistry↗

bioRxiv2025

Identification of High-Risk Cells in Single-Cell Spatially Resolved Transcriptomics Data Using Deep Transfer Learning

SummaryThe examination of high-risk cells and regions in tissue samples from spatially resolved transcriptomics platforms offers meaningful insights into specific disease processes. For existing methods, while cell types or clusters can be identified and associated with disease attributes, individual cells are unable to be associated in the same manner. MethodDiag-nostic Evidence GAuge of Single-cells and spatial transcriptomics (DEGAS), solves the above problem by employing latent representations of gene expression data and domain adaptation to transfer disease attributes from patients to individual cells from single-cell RNA sequencing datasets. In this research, we present and evaluate DEGASs versatility in adapting to data arising from various single-cell spatially resolved transcriptomics (scSRT) platforms. DEGAS successfully identified high-risk cells and regions in liver hepatocellular carcinoma and skin cutaneous melanoma, which were validated through known markers. Additionally, DEGAS was applied to our newly generated Type II Diabetes Xenium dataset revealing high-risk cells within the tissue samples. Availability and ImplementationThe DEGAS software can be accessed at https://github.com/tsteelejohnson91/DEGAS. For the updated smoothing functions and associated codes, visit https://github.com/dchatter04/DEGAS-Spatial-Smoothing. Sources for the datasets reviewed are detailed in their respective sections. A description of some datasets, along with extra tables and figures, is provided in the Supplementary Materials file. Our newly generated Xenium data for Type II Diabetes can be found at https://doi.org/10.7303/syn68699752.

bioinformatics↗

bioRxiv2025

Repurposing of the RIPK1 selective benzooxazepin-4-one scaffold for the development of a type-III LIMK1/2 inhibitor

Benzoxazepinones have been extensively studied as exclusively selective RIP kinase 1 inhibitors. This scaffold binds as a type-III inhibitor targeting the C-out/DFG-out conformation. This inactive conformation results in a large expansion of the kinase back pocket, a conformation that has also been reported for LIM kinases. Scaffold hopping is common in the design of orthosteric kinase inhibitors, but has not been explored in the design of allosteric inhibitors, mainly due to the typically exclusive selectivity of type III inhibitors. Here, we hypothesized that the shared structural properties of LIMKs and RIPKs could lead to novel type III LIMK inhibitors using the benzoxazepinone scaffold. We report the discovery of a novel LIMK1/2 inhibitor that relies on this scaffold-based approach. The discovered compound 10 showed low nanomolar potency on LIMK1/2 and exceptional selectivity, as confirmed by a comprehensive selectivity panel with residual RIPK activity as the only off-target. The study provides one of the few examples for scaffold hopping for type-III inhibitors which are usually associated with exclusive target selectivity. Table of contents (TOC) graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/636296v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@19bad48org.highwire.dtl.DTLVardef@18d8929org.highwire.dtl.DTLVardef@1280043org.highwire.dtl.DTLVardef@1296ddf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Environmental exposures and familial background alter the induction of neuropathology and inflammation after SARS-CoV-2 infection.

AbstractBasal ganglia disease has been reported as a post-infection sequela of several viruses, with documentation of this phenomenon from the H1N1 Spanish flu to the recent COVID-19 (SARS-CoV-2) pandemic. SARS-CoV-2 infection leads to multisystem deficits, including those affecting the nervous system. Here, we investigated whether a SARS-CoV-2 infection alone increases the susceptibility to develop parkinsonian phenotypes in C57BL/6J mice expressing the human ACE2 receptor, or in addition to two well-known toxin exposures, MPTP and paraquat. Additionally, we examined mice carrying a G2019S mutation in the LRRK2 gene. We also examined if vaccination with either an mRNA- or protein-based vaccine can alter any observed neuropathology. We find that the infection with the WA-1/2020 (alpha) or omicron B1.1.529 strains in ACE2 and G2019S LRRK2 mice both synergize with a subtoxic exposure to the mitochondrial toxin MPTP to induce neurodegeneration and neuroinflammation in the substantia nigra. This synergy appears toxin-dependent since we do not observe this following exposure to the direct redox-inducing compound paraquat. This synergistic neurodegeneration and neuroinflammation is rescued in WT mice that were vaccinated using either mRNA- and protein- based vaccines directed against the Spike protein of the SARS-CoV-2 virus. However, in the G2019S LRRK2 mutant mice, we find that only the protein-based vaccine but not the mRNA- based vaccine resulted in a rescue of the SARS-CoV-2 mediated neuropathology. Taken together, our results highlight the role of both environmental exposures and familial background on the development of parkinsonian pathology secondary to viral infection and the benefit of vaccines in reducing these risks.

neuroscience↗

bioRxiv2024

Tropical island endemic birds' demographic responses to climate are modulated by habitat availability and species traits

Climate change and associated habitat fluctuations can expedite the diversification of insular lineages or lead to isolation and extinction. Tropical island birds are a model system to assess responses to climate change owing to their insular nature and ability to diversify rapidly. While there is some understanding of the diversification of tropical island avian clades, we are yet to understand the vulnerability of these species to climate change. Long-term species genetic diversity and historical demography are critical predictors of this. Therefore, we investigated the sensitivity of tropical island endemic birds to climate change and analysed how species traits determine these responses by comparing species traits with demographic histories and paleohabitat fluctuations during the Last Glacial Period (LGP). From publicly available whole genome and paleoclimatic datasets, we reconstructed tropical island endemics past demographic histories (effective population size (Ne)) using Pairwise Sequential Markovian Coalescent (PSMC) (n = 23) and suitable habitat (n = 29) during the LGP and the Holocene. We observed that most species experienced an increase in suitable habitat between the Last Interglacial and the Last Glacial Maximum. However, a concomitant increase in Ne was only observed in the hyper-diverse passerine clade, attesting to their ability to rapidly diversify. Overall, diet specialists and large-bodied species showed a decrease in Ne during the LGP. Our results indicate that species traits dictate tropical island endemics demographic responses to climate, and a plastic response to habitat availability could be a consequence of clades abilities to rapidly occupy new niches and diversify. Further, our analyses revealed that most species entered the Holocene with low effective population sizes. Given that tropical island endemics have small geographic ranges and are groups vulnerable to climate change, special efforts are necessary to conserve them. We recommend that conservation management policies add components like historical demography and species traits while assessing extinction threats for island populations.

evolutionary biology↗

bioRxiv2024

Type-II kinase inhibitors that target Parkinson's Disease-associated LRRK2

Aberrant increases in kinase activity of leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinsons disease (PD). Numerous LRRK2-selective type-I kinase inhibitors have been developed and some have entered clinical trials. In this study, we present the first LRRK2-selective type-II kinase inhibitors. Targeting the inactive conformation of LRRK2 is functionally distinct from targeting the active-like conformation using type-I inhibitors. We designed these inhibitors using a combinatorial chemistry approach fusing selective LRRK2 type-I and promiscuous type-II inhibitors by iterative cycles of synthesis supported by structural biology and activity testing. Our current lead structures are selective and potent LRRK2 inhibitors. Through cellular assays, cryo-electron microscopy structural analysis, and in vitro motility assays, we show that our inhibitors stabilize the open, inactive kinase conformation. These new conformation-specific compounds will be invaluable as tools to study LRRK2s function and regulation, and expand the potential therapeutic options for PD.

cell biology↗

bioRxiv2024

Conserved ancillary residues situated proximally to the VIM-2 active site affect its metallo β-lactamase activity

Verona-integron-metallo-{beta}-lactamase (VIM-2) is one of the most widespread class B {beta}-lactamase, responsible for {beta}-lactam resistance. Although active-site residues help in metal binding, the residues nearing the active-site possess functional importance. Here, to decipher the role of such residues in the activity and stability of VIM-2, the residues E146, D182, N210, S207, and D213 were selected through in-silico analyses and substituted with alanine using site-directed mutagenesis. The effects of substitution mutations were assessed by comparing the changes in the {beta}-lactam susceptibility pattern of E. coli host cells expressing VIM-2 and its mutated proteins. VIM-2_N210A enhanced the susceptibility of the host by [~]4-8 folds against penicillins and cephalosporins while the expression of VIM-2_D182A radically increased the susceptibility of the host. However, expression of VIM-2_E146A reduced the susceptibility of the host by 2-fold. Further, proteins were purified to homogeneity, and VIM_N210A and VIM_D182A displayed reduced thermal stability than VIM-2. Moreover, in vitro catalytic efficiencies of VIM-2_D182A were drastically reduced against all the {beta}-lactams tested whereas the same were moderately reduced for VIM-2_N210A. Conversely, the catalytic efficiency was marginally altered for VIM_E146A. Overall, we infer that both N210A and D182A substitutions negatively affect the performance of VIM-2 by influencing substrate specificity and stability, respectively.

microbiology↗

bioRxiv2024

The asymmetric opening of HIV-1 Env by a potent CD4 mimetic enables anti-coreceptor binding site antibodies to mediate ADCC

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

microbiology↗