Biology subjects

Walsh, M. A.

Publications and source records attributed to Walsh, M. A..

9 recordsLinked to original sources

bioRxiv2024

Crystallographic fragment screening delivers diverse chemical scaffolds for Zika virus NS2B-NS3 protease inhibitor development

The Zika viral protease NS2B-NS3 is essential for the cleavage of viral polyprotein precursor into individual structural and non-structural (NS) proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 47 fragments with diverse scaffolds were identified to bind in the active site of the protease, with another 6 fragments observed in a potential allosteric site. To identify binding sites that are intolerant to mutation and thus suppress the outgrowth of viruses resistant to inhibitors developed from bound fragments, we performed deep mutational scanning of NS2B-NS3 protease. Merging fragment hits yields an extensive set of mergers, defined as synthetically accessible compounds that recapitulate constellations of observed fragment-protein interactions. In addition, the highly sociable fragment hits enable rapid exploration of chemical space via algorithmic calculation and thus yield diverse possible starting points that maximally explore the binding opportunities to NS2B-NS3 protease, facilitating its resistance-resilient antiviral development.

biochemistry↗

bioRxiv2023

Studies on the selectivity of the SARS-CoV-2 papain-like protease reveal the importance of the P2' proline of the viral polyprotein

The SARS-CoV-2 papain-like protease (PLpro) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, releasing the non-structural proteins (nsps) 1-3 that are essential for the coronavirus lifecycle. The LXGG{downarrow}X motif found in pp1a/1ab is crucial for recognition and cleavage by PLpro. We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PLpro. The results reveal how the substrate sequence affects the efficiency of PLpro-catalyzed hydrolysis. In particular, a proline at the P2' position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PLpro catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2' contribute to catalytic efficiency, possibly reflecting the promiscuity of PLpro. The results will help in identifying PLpro substrates and guiding inhibitor design.

bioinformatics↗

bioRxiv2022

xia2.multiplex: a multi-crystal data analysis pipeline

In macromolecular crystallography radiation damage limits the amount of data that can be collected from a single crystal. It is often necessary to merge data sets from multiple crystals, for example small-wedge data collections on micro-crystals, in situ room-temperature data collections, and collection from membrane proteins in lipidic mesophase. Whilst indexing and integration of individual data sets may be relatively straightforward with existing software, merging multiple data sets from small wedges presents new challenges. Identification of a consensus symmetry can be problematic, particularly in the presence of a potential indexing ambiguity. Furthermore, the presence of non-isomorphous or poor-quality data sets may reduce the overall quality of the final merged data set. To facilitate and help optimise the scaling and merging of multiple data sets, we developed a new program, xia2.multiplex, which takes data sets individually integrated with DIALS and performs symmetry analysis, scaling and merging of multicrystal data sets. xia2.multiplex also performs analysis of various pathologies that typically affect multi-crystal data sets, including non-isomorphism, radiation damage and preferential orientation. After describing a number of use cases, we demonstrate the benefit of xia2.multiplex within a wider autoprocessing framework in facilitating a multi-crystal experiment collected as part of in situ room-temperature fragment screening experiments on the SARS-CoV-2 main protease.

molecular biology↗

bioRxiv2021

EFHD1 ablation reduces cardiac mitoflash activation and protects cardiomyocytes from ischemia.

Altered levels of intracellular calcium (Ca2+) is a highly prevalent feature in different forms of cardiac injury, producing changes in contractility, arrhythmias, and mitochondrial dysfunction. In cardiac ischemia-reperfusion injury, mitochondrial Ca2+ overload leads to pathological production of reactive oxygen species (ROS), activates the permeability transition, and cardiomyocyte death. Here we investigated the cardiac phenotype caused by deletion of EF-hand domain-containing protein D1 (Efhd1-/-), a Ca2+-binding mitochondrial protein whose function is poorly understood. Efhd1-/- mice are viable and have no adverse cardiac phenotypes. They feature reductions in basal ROS levels and mitoflash events, both important precursors for mitochondrial injury, though cardiac mitochondria have normal susceptibility to Ca2+ overload. Notably, we also find that Efhd1-/- mice and their cardiomyocytes are resistant to hypoxic injury.

cell biology↗

bioRxiv2021

Discovery of SARS-CoV-2 Mpro Peptide Inhibitors from Modelling Substrate and Ligand Binding

The main protease (Mpro) of SARS-CoV-2 is central to its viral lifecycle and is a promising drug target, but little is known concerning structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of classical molecular mechanics and quantum mechanical techniques, including automated docking, molecular dynamics (MD) simulations, linear-scaling DFT, QM/MM, and interactive MD in virtual reality, to investigate the molecular features underlying recognition of the natural Mpro substrates. Analyses of the subsite interactions of modelled 11-residue cleavage site peptides, ligands from high-throughput crystallography, and designed covalently binding inhibitors were performed. Modelling studies reveal remarkable conservation of hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular at the P2/S2 sites. The binding modes of the natural substrates, together with extensive interaction analyses of inhibitor and fragment binding to Mpro, reveal new opportunities for inhibition. Building on our initial Mpro-substrate models, computational mutagenesis scanning was employed to design peptides with improved affinity and which inhibit Mpro competitively. The combined results provide new insight useful for the development of Mpro inhibitors.

bioinformatics↗

bioRxiv2021

Phytochemical Nrf2 activator attenuates skeletal muscle mitochondrial dysfunction and impaired proteostasis in a preclinical model of musculoskeletal aging

Musculoskeletal dysfunction is an age-related syndrome associated with impaired mitochondrial function and proteostasis. However, few interventions have tested targeting two drivers of musculoskeletal decline. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that stimulates transcription of cytoprotective genes and improves mitochondrial function. We hypothesized daily treatment with a Nrf2 activator in Hartley guinea pigs, a model of age-related musculoskeletal dysfunction, attenuates the progression of skeletal muscle mitochondrial dysfunction and impaired proteostasis, preserving musculoskeletal function. We treated 2-month- and 5-month-old male and female Hartley guinea pigs for 3 and 10 months, respectively, with the phytochemical Nrf2 activator PB125 (Nrf2a). Longitudinal assessments of voluntary mobility were measured using Any-Maze open-field enclosure monitoring. Cumulative skeletal muscle protein synthesis rates were measured using deuterium oxide over the final 30 days of treatment. Mitochondrial oxygen consumption in permeabilized soleus muscles was measured using ex vivo high resolution respirometry. In both sexes, Nrf2a 1) increased electron transfer system capacity; 2) attenuated the disease/age-related decline in coupled and uncoupled mitochondrial respiration; and 3) attenuated declines in protein synthesis in the myofibrillar, mitochondrial, and cytosolic subfractions of the soleus. These improvements were not associated with statistically significant prolonged maintenance of voluntary mobility in guinea pigs. Collectively, these results demonstrate that treatment with an oral Nrf2 activator contributes to maintenance of skeletal muscle mitochondrial function and proteostasis in a pre-clinical model of musculoskeletal decline. Further investigation is necessary to determine if these improvements are also accompanied by slowed progression of other aspects of musculoskeletal decline.

physiology↗

bioRxiv2020

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments was progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

biophysics↗

bioRxiv2019

Structural insights into the molecular mechanisms of the Mycobacterium evolvability factor Mfd

Mfd is a highly conserved ATP dependent DNA translocase that mediates the role of Transcription-Coupled-DNA-Repair(TCR) in bacteria. The molecular mechanisms and conformational remodelling that occurs in Mfd upon ATP binding, hydrolysis, and DNA translocation are poorly defined. Here we report a series of crystal and electron microscopy(EM) structures of Mfd from Mycobacterium tuberculosis (MtbMfd) and Mycobacterium smegmatis Mfd, solved in both the apo and nucleotide-bound states. The structures reveal the mechanism of nucleotide-binding, which lead to the remodeling of the Walker A motif at the catalytic pocket, inducing a flip-flop action of the hinge and flexible linker regions. Specifically, nucleotide binding unlocks the Translocation in RecG motif of the D6-domain to induce a ratchet-like motion. Functional studies of MtbMfd-RNAP complexes show that MtbMfd rescues stalled Transcription Elongation Complexes. We also report negative-stain and cryo-EM single particle reconstructions of MtbMfd higher order oligomer, that reveal an unexpected dodecameric assembly state. Given that Mfd accelerates the evolution of antimicrobial resistance(AMR), our results establish a framework for the design of new \"anti-evolution\" therapeutics to counter AMR.

molecular biology↗

bioRxiv2019

Differential impact on motility and biofilm dispersal of closely related phosphodiesterases in Pseudomonas aeruginosa

Bacteria typically occur either as free-swimming planktonic cells or within a sessile, biofilm mode of growth. In Pseudomonas aeruginosa, the transition between these lifestyles is known to be modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP). We are interested in the control of distinct biofilm-relevant phenotypes in P. aeruginosa through the modulation of intracellular c-di-GMP. Here, we characterise motility and associated biofilm formation and dispersal in two pairs of related multi-domain proteins with putative c-di-GMP turnover domains, selected to contain additional PAS (Per-Arnt-Sim) homology domains known for their ability to process environmental stimuli. The enzymes PA0861 (RbdA) and PA2072 have distinct functions despite their similar domain structures. The {Delta}rbdA deletion mutant showed significantly increased biofilm formation while biofilm formation was impaired in {Delta}PA2072. Using a GFP transcriptional reporter fused to the cyclic di-GMP-responsive cdrA promoter, we show correlation between biofilm phenotype and c-di-GMP levels. Both proteins are shown to play a role in nitric oxide (NO) induced biofilm dispersal. We further studied pseudo-enzymes of similar architecture. PA5017 (DipA) is an inactive cyclase, and PA4959 (FimX) is described here as an inactive cyclase/phosphodiesterase. Loss of swimming and twitching motilities, respectively, is observed in deletion variants, which correlated with NO-induced biofilm dispersal phenotypes, as {Delta}dipA dispersed less well while {Delta}fimX dispersed better than wild type. The study highlights how Pseudomonas differentiates c-di-GMP output - in this case motility - using structurally very similar proteins and underlines a significant role for pseudo-enzymes in motility regulation and associated biofilm dispersal.\n\nImportanceBacterial biofilms exert pervasive economic and societal impact across a range of environmental, engineered and clinical contexts. The secondary messenger cyclic guanosine di-phosphate, c-di-GMP, is known to control the ability of many bacteria to form biofilms. The opportunistic human pathogen Pseudomonas aeruginosa PAO1 has 38 putative enzymes that can regulate c-di-GMP turnover, and these proteins modulate various cellular functions and influence bacterial lifestyle. The specific protein sensory domains and mechanisms of motility that lead to biofilm dispersal remain to be fully understood. Here we studied multi-domain proteins with the PAS (Per-Arnt-Sim) homology domains, these being classic sensors to environmental stimuli. Our study demonstrates the significant roles for the pseudo-enzymes PA4959 (FimX) and PA5017 (DipA) in regulation of biofilm phenotype and motility. Further, enzymes with highly homologous structures, such as PA0861 (RbdA) and PA2072, have almost orthogonal function in biofilm and motility control.

microbiology↗