Biology subjects

Sham, Y. Y.

Publications and source records attributed to Sham, Y. Y..

7 recordsLinked to original sources

bioRxiv2024

Development of non-β-Lactam covalent allosteric inhibitors targeting PBP2a in Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive bacterial pathogen, continues to pose a serious threat to the current public health system in our society. The high level of resistance to {beta}-lactam antibiotics in MRSA is attributed to the expression of penicillin-binding protein 2a (PBP2a), which catalyzes cell wall cross-linking. According to numerous research reports, the activity of the PBP2a protein is known to be regulated by an allosteric site distinct from the active site where cell wall cross-linking occurs. Here, we conducted a screening of 113 compounds containing a 1,3,4-oxadiazole core to design new covalent inhibitors targeting the allosteric site of PBP2a and establish their structural-activity relationship. The stereochemically selective synthesis of sulfonyl oxadiazole compounds identified in the initial screening resulted in a maximum eightfold enhancement in cell inhibition activity. The sulfonyl oxadiazole-based compounds formulated as PEG-based ointments, with low toxicity test results on human cells (CC50: >78M), demonstrated potent antimicrobial effects not only in a mouse skin wound infection model but also against oxacillin-resistant clinical isolate MRSA (IC50 {approx} 1M), as evidenced by the results. Furthermore, additional studies utilizing LC-MS/MS and in-silico approaches clearly support the allosteric site covalent binding mechanism through the nucleophilic aromatic substitution (SNAr) reaction, as well as its association with the closure of the major active site of PBP2a.

microbiology↗

bioRxiv2024

Carbapenem-induced β-lactamase-isoform expression trends in Acinetobacter baumannii

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an urgent bacterial threat to public health, with only a few treatment options and a >50% fatality rate. Although several resistance mechanisms are understood, the appearance of these mutations is generally considered stochastic. Recent reports have, however, begun to challenge this assumption. Here, we demonstrate that independent samples of Ab, exposed to different carbapenems with escalating concentrations, show concentration- and carbapenem-dependent trends in {beta}-lactamase-isoform expression. This result, based on the isoforms identified through label-free-quantification LC-MS/MS measurements of cell-free, gel-separated {beta}-lactamases, suggests that the appearance of antibiotic resistance may be somewhat non-stochastic. Specifically, several minor AmpC/ADC {beta}-lactamase-isoforms were found to exhibit both dose- and carbapenem-dependent expression, suggesting the possibility of non-stochastic mutations. Additionally, these also have high sequence similarity to major expressed isoforms, indicating a potential path over which resistance occurred in independent samples. Antibiotic resistance maybe somewhat antibiotic-directed by a hitherto unknown mechanism and further investigation may lead to new strategies for mitigating antibiotic resistance. TeaserThe emergence of antibiotic-resistant {beta}-lactamase proteins from mutations may exhibit patterns based on specific antibiotics.

microbiology↗

bioRxiv2022

Kinetic Characterization and Computational Modeling of the Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for a GT-B Glycosyltransferase

Glycosyltransferases (GTs) are enzymes that are uniquely adapted to promote the formation of a glycosidic bond between a sugar molecule and a wide variety of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (L-glycero-D-manno-heptose; Hep) onto a lipid anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A or HLA). LPS plays a key role in Gram-negative bacterial sepsis as a stimulator of the human immune response and has been used as an adjuvant in vaccines. As such, ongoing efforts towards inhibition of LPS biosynthetic enzymes to aid development of novel antimicrobial therapeutics has driven significant effort towards the characterization of these enzymes. Three heptosyltransferases are involved in the inner-core biosynthesis, with E. coli HepII being the last to be quantitatively characterized in vivo, as described herein. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared by HepI to include sugar donor promiscuity, and sugar acceptor induced secondary structural changes which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates, with an insensitivity to the number of acyl chains on the sugar acceptor. Additionally, a structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe potentially important substrate-protein contacts and revealed the potential of Tryptophan (Trp) residues responsible for reporting on ligand binding. As was previously described for HepI, Tryptophan fluorescence in HepII allowed observation of substrate induced changes in Trp fluorescence intensity which enabled determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the Heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent and specific inhibitors for this family of enzymes.

biochemistry↗

bioRxiv2021

Discovery of Aminoglycosides as first in class, nanomolar inhibitors of Heptosyltransferase I

A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years and while many have designed novel small-molecule inhibitors, these compounds lack the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. With the help of molecular dynamic simulations, aminoglycoside antibiotics were shown to be putative inhibitors for HepI and in this study, they were experimentally determined to be the first in-class nanomolar inhibitors of HepI with the best inhibitor demonstrating a Ki of 600 +/- 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and MD simulations were used to corroborate kinetic experimental findings. Kinetic analysis methods include Lineweaver-Burk, Dixon, Cornish-Bowden and Mixed-Model of Inhibition which allowed for unambiguous assignment of inhibition mechanism for each inhibitor. In this study, we show that neomycin and kanamycin b are competitive inhibitors against the sugar acceptor substrate while tobramycin exhibits a mixed inhibitory effect and streptomycin is non-competitive. MD simulations also allowed us to suggest that the inhibitors bind tightly and inhibit catalytic dynamics due to a major desolvation penalty of the enzyme active site. While aminoglycosides have long been known as a class of potent antibiotics, they also have been scientifically shown to impact cell membrane stability, and we propose that inhibition of HepI contributes to this effect by disrupting lipopolysaccharide biosynthesis.

biochemistry↗

bioRxiv2021

Ligand Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase:Molecular Dynamic Simulations of Heptosyltransferase I Apo, Binary and Ternary Complexes

Understanding the dynamical motions and ligand recognition motifs of specific glycosyltransferase enzymes, like Heptosyltransferase I (HepI), is critical to discerning the behavior of other carbohydrate binding enzymes. Prior studies in our lab demonstrated that glycosyltransferases in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conservation of both structure and dynamical motions, despite low sequence conservation, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through a series of 100 nanosecond Molecular Dynamics simulations of HepI in apo enzyme state, and also in the binary and ternary complexes with the native substrates/products. Ligand free energy analysis allowed determination of an anticipated enzymatic path for ligand binding and release. Principle component, dynamic cross correlation and network analyses of the simulation trajectories revealed that there are not only correlated motions between the N- and C-termini, but also that residues within the N-terminal domain communicate via a path that includes substrate proximal residues of the C-terminal domain. Analysis of structural changes, energetics of substrate/products binding and changes in pKa have elucidated a variety of inter- and intradomain interactions that are critical for catalysis. These data corroborate and allow visualization of previous experimental observations of protein conformational changes of HepI. This study has provided valuable insights into the regions involved in HepI conformational rearrangement upon ligand binding, and are likely to enhance efforts to develop new dynamics disrupting enzyme inhibitors for GT-B structural enzymes in the future.

biochemistry↗

bioRxiv2020

Conserved conformational hierarchy across functionally divergent glycosyltransferases of the GT-B structural superfamily as determined from microsecond molecular dynamics

It has long been understood that some proteins to undergo conformational transitions enroute to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site is necessary for the protein to crystalize in the closed conformation. Herein we describe microsecond molecular dynamics simulations of two evolutionarily unrelated glycosytransferases, HepI and GtfA. Simulations were performed using these proteins in the open and closed conformations, (respectively,) and we sought to identify the major dynamical modes and communication networks which allow conformational transition between the open and closed structures. We provide the first reported evidence (within the scope of our experimental parameters) that conformational hierarchy/directionality of the interconversion between open and closed conformations is a conserved feature of enzymes of the same structural superfamily. Additionally, residues previously identified to be important for substrate binding in HepI were shown to have strong negative correlations with non-ionizable residues distal to the active site. Mutagenesis of these residues produced mutants with altered enzymatic efficiency exhibiting lower Km values, while the kcat is effectively unchanged. The negatively correlated motions of these residues are important for substrate binding and forming the Michaelis complex, without impacting the activation barrier for catalysis. This suggests that in the bi-domain HepI, the enzyme dynamics did not impact the transition state stabilization or chemistry, but rather earlier steps along the reaction coordinate, leading to the reorganization of the active site electrostatic environment required for catalysis.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsGTglycosyltransferaseHepIheptosyltransferase IheptoseL-glycero-D-manno-heptoseADP-heptose or ADPHADP-L-glycero-D-manno-heptoseLPSlipopolysaccharide;ODLAO-deacylated E. coli Kdo2-lipid A;Trptryptophan;Proproline;Glyglycine;Argarginine;Lyslysine;CDcircular dichroism;MDMolecular Dynamics;PDBProtein Data BankPCAPrincipal Component Analysis,DCCDynamic Cross CorrelationView Full Text

biophysics↗

bioRxiv2019

CD200 immune checkpoint reversal at the site of tumor vaccine inoculation: A novel approach to glioblastoma immunotherapy

PurposeAdvances in immunotherapy have revolutionized care for some cancer patients. However, current checkpoint inhibitors are associated with significant toxicity and yield poor responses for patients with central nervous system tumors, calling into question whether cancer immunotherapy can be applied to glioblastoma multiforme. We determined that targeting the CD200 activation receptors (CD200AR) of the CD200 checkpoint with a peptide inhibitor overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the peptide inhibitor in a trial in companion dogs with spontaneous high-grade glioma; adding the peptide to autologous tumor lysate vaccines significantly increased overall survival relative to tumor lysate alone (median survival of 12.7 and 6.36 months, respectively).\n\nExperimental design: This study was developed to elucidate the mechanism of the CD200ARs and develop a humanized peptide inhibitorWe developed macrophage cell lines with each of four CD200ARs knocked out to determine their binding specificity and functional responses. Using proteomics, we developed humanized peptide inhibitors to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in CD14+ cells. GMP-grade peptide was further validated for activity.\n\nResultsWe demonstrated that the peptide specifically targets the CD200AR complex to induce an immune response. Moreover, we developed and validated our humanized peptides for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhancing an antigen-specific response. We also determined that the use of the peptide downregulated the expression of CD200 inhibitory and PD-1 receptors.\n\nConclusionThese results support consideration of a CD200 peptide ligand as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.\n\nTranslational relevanceThis report evaluates the ability to modulate the CD200 immune checkpoint by employing synthetic peptides directed as ligands to its paired immune activation receptor. We previously reported the presence of CD200 in the sera and tumor vasculature of patients with glioblastoma multiforme (GBM). We have also shown that a canine CD200 activation receptor ligand extends the lives of companion dogs with high grade glioma. The data we present here show that the human peptide ligand (hCD200ARL) directed to the CD200 activation receptor on CD14+ cells activates immune upregulation through induction of a cytokine response and dendritic cell differentiation. In addition, hCD200ARL downregulates the inhibitory CD200 and PD-1 receptors. These findings provide a basis to evaluate hCD200ARL as a novel immune therapy for patients with GBM. Downregulation of PD-1 suggests that hCD200ARL may also obviate the need for PD1 and PD-L1 directed therapies for GBM and other malignancies.

cancer biology↗