Biology subjects

Rahman, K. M.

Publications and source records attributed to Rahman, K. M..

11 recordsLinked to original sources

bioRxiv2026

Structural basis of ligand-selective transcriptional activation in the MerR-family antibiotic resistance regulator AlbA

Multidrug-resistant pathogens demand novel resistance-breaking strategies. The oligoarylamide albicidin and pyrrolobenzodiazepines (PBDs) are potent antibacterials with unrelated scaffolds, yet both are neutralized by albA gene products in many Gram-negatives: AlbA, an albicidin-responsive self-upregulating MerR-family factor, and the smaller AlbAS, its unique ligand binding domain (LBD) alone. How AlbA couples ligand sensing to transcriptional activation has remained elusive. Here, using an integrative multidisciplinary approach, we define this mechanism and show that albicidin and PBDs elicit different responses. Crystal structures reveal C8-linked PBDs bound to AlbAS at the N- and C-terminal subdomains (NTD, CTD) of the central tunnel in poses dictated by C8-tail chemistry. This plasticity sequesters diverse PBDs with nanomolar affinities. Cryo-EM shows AlbA as an autoinhibited dimer in which a reciprocal arm closes the NTD end of the partner's tunnel, restricting PBD binding to the CTD while the DNA binding domains (DBDs) are mobile. Albicidin binding is incompatible with this arrangement, whereas PBDs, as CTD plugs, do not perturb the native equilibrium between autoinhibited and promoter-competent states. We visualized the latter by cryo-EM in the RNAP-DNA-AlbA complex. By blocking albicidin-mediated enhancement in vitro, PBDs act as resistance-breaking partners for albicidin and a route to overcoming albA-dependent resistance to oligoarylamide antibiotics.

biochemistry↗

bioRxiv2026

Evaluation of selectively-activatable, caged fluorescent probes as species selective markers for beta-alanine aminopeptidase positive bacterial species

Aminopeptidases are widely distributed in bacteria, but outside of a few model strains, their function is largely unexplored. Focussing on beta-alanine aminopeptidase activity, a new series of selectively-activatable, caged fluorescent probes were designed and synthesised. A beta alanine amino acid was coupled to resorufin or 7-hydroxycoumarin via a self-imolative linker, such that amino acid removal led to gain of fluorescence. These were used to probe selectivity and specificity of probe activation, against a range of priority drug-resistant pathogens. When added to bacterial growth curves run in Muller Hinton broth, these probes allowed essentially real time fluorescence measurement of activation by bacterial species, modelled on the standard microbroth dilution method. Activation was observed for all Pseudomonas aeruginosa and Burkholderia spp strains tested. Selective activation was seen for Ochrabactrum species, with the probe activated by O.anthropii (2/4 strains) but not O.intermedium and strain-specific activation was seen for some isolates of Serratia marcescens (2/4 strains). No activation was observed in any isolates of Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii or Staphylococcus aureus or Eneterocccus faecium/faecalis PAO1 transposon mutants in the putative beta-alanine aminopeptidase gene (annotated as bapF or dmpA; PW3678) showed no activation of the probe in growth assays, confirming the specificity of the probe for beta-alanine aminopeptidase. Transposon mutants in other aminopeptidase genes, including those encoded by pepN, PepP and the prolyl aminopeptidase gene had no effect on probe activation in PAO1. Based on the operon structure in PA01, transposon mutants in two adjacent genes were also tested for probe activation. Mutants in both a putative transcriptional regulator (PW3674) and a predicted amino acid permease (PW3676) retained their ability to activate the beta-alanine probes with activation significantly higher than the wild type, when assessed by the total fluorescence yield after 10 hours growth. This points to both redundancy in permease function and perhaps the presence of a feedback regulatory mechanism controlling beta alanine aminopeptidase activity in P.aeruginosa. Given that the operon structure is conserved in other species, this may point to a common mechanism of beta alanine aminopeptidase function, perhaps related to exploiting beta-alanine containing peptides in certain environmental niches.

microbiology↗

bioRxiv2026

Discovery of a pathway-selective platelet P2Y1R inverse agonist that suppresses inflammation while preserving hemostasis

The platelet P2Y1 receptor (P2Y1R) is necessary for inflammation, signalling via Rho-GTPase pathways to elicit functions that are distinct from aggregation (PLC-dependent canonical signalling pathway). Whether these distinct platelet inflammatory functions can be selectively suppressed to preserve hemostasis through the rational design of P2Y1R antagonists has not been explored. In silico molecular docking analysis examined biased nucleotide interactions within the P2Y1R binding pocket. The identified possible key amino acid residues guided rational design to synthesize compounds for pathway selective inhibition, evolving from nucleotide to non-nucleotide structures. The nucleotide analogue KMR-82-13 was predicted to engage distinct regions of the binding pocket and selectively inhibited platelet chemotaxis while preserving aggregation. These findings informed the design of a non-nucleotide compound KSN-159-27, aiming to retain key KMR-82-13-like interactions while improving drug-like properties. Docking and molecular dynamics simulation supported a stable but dynamic binding mode for KSN-159-27 within the P2Y1R pocket, consistent with pathway-selective inhibition. KSN-159-27 displayed characteristics of a pathway selective inverse agonist at P2Y1R towards G12/13-mediated pathways, but not those associated by Gq activation in P2Y1R-transfected HEK293T cells. KSN-159-27 showed functionally selective inhibition for platelet P2Y1R-mediated functions. In vivo, KSN-159-27 suppressed inflammatory cell recruitment, whilst preserving bleeding time and ADP-induced thromboembolic responses, in contrast to the neutral P2Y1R antagonist MRS2500. This first demonstration for the rational design of a pathway selective inverse agonist at platelet P2Y1Rs has significant implications for novel therapeutic strategies developed to safely target platelet activation during inflammation, in contrast to current anti-platelet drugs used in the prevention of thrombosis. Key PointsO_LIBiased inverse platelet P2Y1R agonists selectively supress inflammation whilst preserving hemostasis and the ability of platelets to aggregate. C_LIO_LIBiased inverse agonism selectively inhibited P2Y1R G12/13 (Rho-GTPAse functions) but not Gq activities (PLC functions). C_LI

pharmacology and toxicology↗

bioRxiv2024

Construction of a drug sensitised Candida auris strain

Candida auris is an emerging pathogenic fungus causing outbreaks of invasive disease in healthcare facilities worldwide with the majority of clinical isolates demonstrating intrinsic resistance to multiple drug classes. Therefore, there is a pressing need for new antifungals, but screening in wild-type C. auris strains may miss scaffolds which would be good subjects for med-chem projects as antifungals. Work presented here indicates that deletion of CDR1 and MDR1 in C. auris, respectively encoding an ATP-binding Cassette and a Major Facilitator Superfamily efflux pump, generates a strain that is hypersensitised to drugs and can act as a platform for antifungal drug screening. The cdr1{Delta}mdr1{Delta} mutant was 64- and 8- times more sensitive to fluconazole and voriconazole, respectively, compared with the wild-type strain. In a pilot study using a 2,000 compound library, use of the cdr1{Delta}mdr1{Delta} mutant identified over twice as many compounds compared to wild-type. When assessing the capacity to form biofilms, there was a modest difference in biomass between the wild-type and cdr1{Delta}mdr1{Delta} mutant, but there was little to no statistically significant difference observed when using a colorimetric dye that measured metabolic activity. Little to no statistically significant difference was observed between wild-type and the efflux pump mutant in virulence, as judged by the larval Galleria mellonella infectivity assay. Accordingly, the cdr1{Delta}mdr1{Delta} mutant will be useful when screening for drugs that limit formation of biofilms or limit fungal virulence, as these two properties are unaffected when drug efflux is compromised.

microbiology↗

bioRxiv2024

SARS-CoV-2 infection enhancement by Amphotericin B: Implications for disease management

Severe coronavirus disease 2019 (COVID-19) patients who require hospitalisation are at high risk of invasive pulmonary mucormycosis. Amphotericin B (AmB), which is the first line therapy for invasive pulmonary mucormycosis, has been shown to promote or inhibit replication of a spectrum of viruses. In this study, we first predicted that AmB and Nystatin had strong interactions with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins using in silico screening, indicative of drugs with potential therapeutic activity against this virus. Subsequently, we investigated the impact of AmB, Nystatin, Natamycin, Fluconazole and Caspofungin on SARS-CoV-2 infection and replication in vitro. Results showed that AmB and Nystatin actually increased SARS-CoV-2 replication in Vero E6, Calu-3 and Huh7 cells. At optimal concentrations, AmB and Nystatin increase SARS-CoV-2 replication by up to 100- and 10-fold in Vero E6 and Calu-3 cells, respectively. The other antifungals tested had no impact on SARS-CoV-2 infection in vitro. Drug kinetic studies indicate that AmB enhances SARS-CoV-2 infection by promoting viral entry into cells. Additionally, knockdown of genes encoding for interferon-induced transmembrane (IFITM) proteins 1, 2, and 3 suggests AmB enhances SARS-CoV-2 cell entry by overcoming the antiviral effect of the IFITM3 protein. This study further elucidates the role of IFITM3 in viral entry and highlights the potential dangers of treating COVID-19 patients, with invasive pulmonary mucormycosis, using AmB. ImportanceAmB and Nystatin are common treatments for fungal infections but were predicted to strongly interact with SARS-CoV-2 proteins, indicating their potential modulation or inhibition against the virus. However, our tests revealed that these antifungals, in fact, enhance SARS-CoV-2 infection by facilitating viral entry into cells. The magnitude of enhancement could be up to 10- or 100-fold depending on cell lines used. These findings indicate that AmB and Nystatin have the potential to enhance disease when given to patients infected with SARS-CoV-2 and therefore should not be used for treatment of fungal infections in active COVID-19 cases.

microbiology↗

bioRxiv2024

C7-substituted Quinolines as Potent Inhibitors of AdeG Efflux Pumps in Acinetobacter baumannii

Efflux, mediated by a series of multidrug efflux pumps, is a major contributor to antibiotic resistance in Gram-negative bacteria. Efflux pump inhibitors (EPIs), which can block efflux, have the potential to be used as adjuvant therapies to re-sensitize bacteria to existing antibiotics. In this study, 36 quinoline-based compounds were synthesized as potential EPIs targeting Resistance Nodulation Division (RND) family pumps in the multidrug-resistant pathogen Acinetobacter baumannii . In A. baumannii strains with overexpressed AdeFGH (chloramphenicol-adapted) and AdeABC (AYE, Ab5075-UW), these compounds enhanced Hoechst dye accumulation, indicating general efflux inhibition, and potentiated chloramphenicol which is an AdeG substrate. The research focused on two generations of quinoline compounds, with modifications at the C-7 position of first-generation compounds to improve hydrophobic interactions with the Phe loop in the AdeG efflux pump, to generate second-generation compounds. The modified quinolines showed strong pump inhibition and significant chloramphenicol potentiation, with MIC reductions of 4- to 64-fold. Notably, compounds 1.8 and 3.8 exhibited the highest inhibitory activity, while compounds 1.3 and 3.3 showed up to 64-fold potentiation, highlighting the importance of specific structural features at the C-7 position for efflux pump inhibition. The study also revealed selective inhibition of AdeFGH over AdeABC, with no potentiation observed for gentamicin, showing the specificity of these quinoline- based inhibitors. Importantly, the compounds showed no toxicity in a Galleria mellonella model at a 50 mg/kg dose level, highlighting their suitability as potential antibiotic adjuvants for combating bacterial resistance.

microbiology↗

bioRxiv2023

Alignment Free Phylogeny Construction Using Maximum Likelihood Using k-mer Counts

Estimating phylogenetic trees from molecular data often involves first performing a multiple sequence alignment of the sequences and then identifying the tree that maximizes likelihood computed under a model of nucleotide substitution. However, sequence alignment is computationally challenging for long sequences, especially in the presence of genomic rearrangements. To address this, methods for constructing phylogenetic trees without aligning the sequences i.e. alignment-free methods have been proposed. They are generally fast and can be used to construct phylogenetic trees of a large number of species but they primarily estimate phylogenies by computing pairwise distances and are not based on statistical models of molecular evolution. In this paper, we introduce a model for k-mer frequency change based on a birth-death-migration process which can be used to estimate maximum likelihood phylogenies from frequencies of k-mers in genomic sequences of species in an alignment-free approach. Experiments on real and simulated data demonstrate the efficacy of the model for likelihood based alignment-free phylogeny construction.

evolutionary biology↗

bioRxiv2023

Interaction of amisulpride with GLUT1 at the blood-brain barrier. Relevance to Alzheimer's disease

Blood-brain barrier (BBB) dysfunction may be involved in the increased sensitivity of Alzheimers disease (AD) patients to antipsychotics, including amisulpride. Studies indicate that antipsychotics interact with facilitated glucose transporters (GLUT), including GLUT1, and that GLUT1 BBB expression decreases in AD. We tested the hypotheses that amisulpride (charge: +1) interacts with GLUT1, and that BBB transport of amisulpride is compromised in AD. GLUT1 substrates and inhibitors, and GLUT-interacting antipsychotics were identified by literature review and their physicochemical characteristics summarised. Interactions between amisulpride, and GLUT1 were studied using in silico approaches and the human cerebral endothelial cell line, hCMEC/D3. Brain distribution of [3H]amisulpride was determined using in situ perfusion in wild type (WT) and 5xFamilial AD (5xFAD) mice. With transmission electron microscopy (TEM) we investigated brain capillary degeneration in WT and 5xFAD mice, and human samples. Western blots determined BBB transporter expression in mouse and human. Literature review revealed that, although D-glucose has no charge, charged molecules can interact with GLUT1. GLUT1 substrates are smaller (184.95{+/-}6.45g/mol) than inhibitors (325.50{+/-}14.40g/mol), and GLUT-interacting antipsychotics (369.38{+/-}16.04). Molecular docking showed beta-D-glucose (free energy binding: -15.39kcal/mol) and amisulpride (-29.04kcal/mol) interact with GLUT1. Amisulpride did not affect [14C]D-glucose accumulation in hCMEC/D3. 5xFAD mice showed increased brain [3H]amisulpride uptake, and no cerebrovascular space changes compared to WT. TEM revealed brain capillary degeneration in human AD. There was no significant effect of AD on mouse GLUT1 and P-gp BBB expression, and in human GLUT1 expression. In contrast, caudate P-glycoprotein expression was decreased in human AD capillaries versus controls. This study provides new details about the BBB transport of amisulpride, evidence that amisulpride interacts with GLUT1, and that BBB transporter expression is altered in AD. This suggests that antipsychotics exacerbate the cerebral hypometabolism in AD. Further research into the mechanism of amisulpride transport by GLUT1 is important for improving antipsychotics safety.

neuroscience↗

bioRxiv2022

Stimulation of platelet P2Y1 receptors by differentendogenous nucleotides leads to functional selectivity via biased signalling.

Background and PurposePlatelet function during inflammation is dependent on activation by endogenous nucleotides. Non-canonical signalling via the P2Y1 receptor is important for these non-thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD+, ADP-ribose, and Up4A on platelet functions contributing to inflammation or haemostasis. Experimental ApproachPlatelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD+, ADP-ribose or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y1 receptors was then assessed. Key ResultsPlatelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD+, ADP-ribose, and Up4A. However, these endogenous nucleotides induced P2Y1-dependent platelet chemotaxis, an effect that required RhoA and Rac-1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket by Ap3A, NAD+, ADP-ribose or Up4A compared to ADP. Conclusion and ImplicationsPlatelet function (aggregation vs motility) can be differentially modulated by biased-agonist activation of P2Y1 receptors. This may be due to the character of the ligand-binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant-platelet drugs.

pharmacology and toxicology↗

bioRxiv2020

Integron Gene Cassettes Harboring Novel Variants of D-Alanine-D-Alanine Ligase Confer High-level Resistance to D-Cycloserine

During a PCR-based screen of antibiotic resistance genes (ARGs) associated with integrons in saliva-derived metagenomic DNA of healthy human volunteers, two novel variants of genes encoding a D-alanine-D-alanine ligase (ddl6 and ddl7) located within gene cassettes in the first position of a reverse integron were identified. Treponema denticola was identified as the likely host of the ddl cassettes. Both ddl6 and ddl7 conferred high level resistance to D-cycloserine when expressed in Escherichia coli with ddl7 conferring four-fold higher resistance to D-cycloserine compared to ddl6. A SNP was found to be responsible for this difference in resistance phenotype between both ddl variants. Molecular dynamics simulations were used to explain the mechanism of this phenotypic change at the atomic scale. A hypothesis for the evolutionary selection of ddl containing integron gene cassettes is proposed, based on molecular docking of plant metabolites within the ATP and D-cycloserine binding pockets of Ddl. Significance StatementThe drivers of antimicrobial resistance are varied and numerous. One such hypothesis is that compounds not normally considered to be antibiotics may be able to select for genes that can provide resistance to conventional antibiotics. Here we provide evidence that dietary flavonoids are likely to provide the selective pressure for two novel variants of the ddl gene, encoding D-alanine-D-alanine ligases, to be maintained as the first gene cassette of a reverse integron detectable in the human oral cavities from both the UK and Bangladesh. We show that Ddl is functional, able to confer resistance to D-cycloserine and that the dietary flavonoids quercetin and apigenin are likely able to compete with both ATP and D-cycloserine within their Ddl binding sites.

microbiology↗

bioRxiv2019

Region-specific blood-brain barrier transporter changes leads to increased sensitivity to amisulpride in Alzheimer’s disease

Research into amisulpride use in Alzheimers disease (AD) implicates blood-brain barrier (BBB) dysfunction in antipsychotic sensitivity. Solute carrier function in AD has not been widely studied. This study tests the hypothesis that organic cation transporters contribute to the BBB delivery of antipsychotics and is disrupted in AD.\n\nIn vitro BBB studies indicated that [3H]amisulpride and [3H]haloperidol were transported by OCT1. Amisulpride also utilized PMAT. Molecular docking predicted that amisulpride and haloperidol are OCT1, PMAT and MATE1 substrates, and amisulpride is not a P-gp substrate. Amisulpride brain uptake increased in 3xTgAD compared to wildtype mice. PMAT and MATE1 expression was reduced in brain from AD patients compared to controls. The increased sensitivity of Alzheimers patients to amisulpride is related to previously unreported changes in OCT1, PMAT and MATE1 function/expression at the BBB. Dose adjustments may be required for drugs that are substrates of these transporters when prescribing for AD patients.

neuroscience↗