Scholarly Works, Virginia Tech Center for Drug Discovery

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Structure-activity relationship study of benserazide derivatives as PilB inhibitors
Quinlan, Joseph E.; Soleymani, Ghazal; Shimozono, Tori M.; Yang, Zhaomin; Santos, Webster L. (Royal Society of Chemistry, 2025-06-04)
Antimicrobial resistance is an imminent health threat worldwide. Development of alternative treatments for drug-resistant microbes is of paramount importance. Targeting virulence factors, such as the type IV pilus construction enzyme PilB, is a strategy of treatment. Recently, we reported the discovery of a potent inhibitor of PilB, the FDA approved drug benserazide (IC50 = 3.68 μM). Herein, we report the structure-activity relationship profiling of benserazide analogues and identify key moieties that enable PilB inhibition. We found that bis-hydroxyl groups on the ortho position of the aryl ring, a rigid imine, and exchange of the serine for a thiol have resulted in marked improvement in potency. Our studies identified 11c as a PilB inhibitor with an IC50 of 580 nM and selectivity for PilB over an unrelated ATPase, apyrase. These compounds provide the chemical tools to validate virulence factors as antibacterial mechanisms of action.
Tuning Polyacrylate Composition to Recognize and Modulate Fluorescent Proteins
Gomez, Darwin C.; Seth, Swarnadeep; Mondal, Ronnie; Koehler, Stephen J.; Baker, Jared G.; Plate, Charles; Anderson, Ian C.; Smith, Mikayla R.; Gloriod, Joey; Gunter, Morgan; Welborn, Valerie Vaissier; Deshmukh, Sanket A.; Figg, C. Adrian (Wiley-VCH, 2026-01-09)
Molecular definition is usually regarded as a prerequisite to achieve protein recognition and functional modulation, particularly for macromolecular interactions. Herein, we report that polymers with specific combinations of monomers arranged into random sequences [random hetero oligomers (RHOs)] can selectively bind to a model protein. Using green fluorescent protein (GFP) as a target, polyacrylates were developed that bound with nanomolar affinity and enhanced fluorescence by >100%. Purification of the polymerization product revealed subpopulations of compositions with distinct affinities and selectivity for GFP over a competing protein. Experimental and computational binding analyses confirmed that there are distinct RHO–GFP interactions, which are influenced by RHO chemical composition. These findings show that sequence-defined structures are not a prerequisite for selective protein recognition. Synthetic polymers can instead serve as scalable, tunable platforms for molecular recognition—representing a significant leap towards next-generation sensing, therapeutic, responsive, and catalytic materials in domains previously dominated by biologics or complex peptide scaffolds.
Integration of experimental data and use of automated fitting methods in developing protein force fields
Polêto, Marcelo D.; Lemkul, Justin A. (2022-03)
The development of accurate protein force fields has been the cornerstone of molecular simulations for the past 50 years. During this period, many lessons have been learned regarding the use of experimental target data and parameter fitting procedures. Here, we review recent advances in protein force field development. We discuss the recent emergence of polarizable force fields and the role of electronic polarization and areas in which additive force fields fall short. The use of automated fitting methods and the inclusion of additional experimental solution data during parametrization is discussed as a means to highlight possible routes to improve the accuracy of force fields even further.
Monomeric and dimeric states of human ZO1-PDZ2 are functional partners of the SARS-CoV-2 E protein
Giacon, Noah; Lo Cascio, Ettore; Davidson, Darcy S.; Polêto, Marcelo D.; Lemkul, Justin A.; Pennacchietti, Valeria; Pagano, Livia; Zamparelli, Carlotta; Toto, Angelo; Arcovito, Alessandro (Elsevier, 2023-05)
The Envelope (E) protein of SARS-CoV-2 plays a key role in virus maturation, assembly, and virulence mechanisms. The E protein is characterized by the presence of a PDZ-binding motif (PBM) at its C-terminus that allows it to interact with several PDZ-containing proteins in the intracellular environment. One of the main binding partners of the SARS-CoV-2 E protein is the PDZ2 domain of ZO1, a protein with a crucial role in the formation of epithelial and endothelial tight junctions (TJs). In this work, through a combination of analytical ultracentrifugation analysis and equilibrium and kinetic folding experiments, we show that ZO1-PDZ2 domain is able to fold in a monomeric state, an alternative form to the dimeric conformation that is reported to be functional in the cell for TJs assembly. Importantly, surface plasmon resonance (SPR) data indicate that the PDZ2 monomer is fully functional and capable of binding the C-terminal portion of the E protein of SARS-CoV-2, with a measured affinity in the micromolar range. Moreover, we present a detailed computational analysis of the complex between the C-terminal portion of E protein with ZO1-PDZ2, both in its monomeric conformation (computed as a high confidence AlphaFold2 model) and dimeric conformation (obtained from the Protein Data Bank), by using both polarizable and nonpolarizable simulations. Together, our results indicate both the monomeric and dimeric states of PDZ2 to be functional partners of the E protein, with similar binding mechanisms, and provide mechanistic and structural information about a fundamental interaction required for the replication of SARS-CoV-2.
Drude polarizable force field for phosphorylated polypeptides and proteins
Gil Pineda, Laura I.; Miller, Candace J.; MacKerell, Alexander D. Jr.; Lemkul, Justin A. (Elsevier, 2025-10-22)
Phosphorylation is a ubiquitous posttranslational modification used across all domains of life to regulate protein stability, folding, subcellular localization, function, and activity. The most common targets of protein phosphorylation are the amino acids serine, threonine, and tyrosine. The considerable change in electrostatic character of these residues upon phosphorylation, from polar neutral to strongly negatively charged at physiological pH, implies a major change in biophysical properties of these residues. Modeling phosphorylated residues in molecular dynamics simulations is a challenge given their highly charged nature, and the development of polarizable force field parameters is an important task for the extension of protein force fields. Here, we present the parametrization and validation of force field parameters for phosphorylated serine, threonine, and tyrosine in all protonation states (neutral, monoanionic, and dianionic). We targeted a range of quantum mechanical properties for electrostatic and dihedral parameter fitting and subsequently validated the force field model in the context of two full-length proteins (ERK2 and WNK1) and short polypeptides. We found that the phosphorylated amino acids maintained the expected strong interactions with lysine and arginine residues, rigidifying loops in pERK2 and pWNK1. In the short polypeptides, phosphorylation of serine and threonine reduced disorder and promoted the formation of α-helical turns, in agreement with previous findings. The present force field is compatible with the Drude protein force field and expands the coverage of chemical space to include these important chemical entities for future investigations of protein structure and function, particularly for conformational changes that arise in intrinsically disordered proteins upon phosphorylation.
Running Gaussian-accelerated Molecular Dynamics Simulations in NAMD [Article v1.0]
Michel, Haley M.; Polêto, Marcelo D.; Lemkul, Justin A. (University of Colorado at Boulder, 2025-07)
Gaussian-accelerated molecular dynamics (GaMD) simulations are an advanced technique that enhances the sampling of configurational space by applying biasing potentials that reduce energy barriers, enabling faster exploration of the free energy landscape. This tutorial demonstrates the application of GaMD to the alanine dipeptide, serving as an accessible model system, and guides users through all GaMD simulation stages: conventional MD, GaMD equilibration, GaMD production, and reweighting. Users will gain practical insights into the preparation of input files, monitoring of GaMD convergence, and analysis of free energy profiles using PyReweighting. We make a particular effort to connect the underlying theory with the GaMD workflow. This tutorial is intended for users with prior molecular dynamics experience, Linux and command-line navigation, and with basic Python knowledge. The step-by-step instructions and accompanying scripts aim to streamline the GaMD workflow, making it accessible for the broader research community to explore enhanced sampling for a range of biomolecular systems.
Weighted Ensemble Simulations With the Drude Polarizable Model
Polêto, Marcelo D.; da Silva, Gabriel Monteiro; Rubenstein, Brenda M.; Lemkul, Justin A. (Wiley, 2025-11-15)
Enhanced sampling methods have become powerful techniques to investigate complex systems and rare molecular events by facilitating greater access to configurational space. In parallel, the continuous development of polarizable force fields augments such techniques, allowing the investigation of the role of electronic polarization in transitional barriers that might be overlooked in nonpolarizable simulations. Together, improved sampling methods and polarizable force fields should accelerate computational discoveries. Here, we present an implementation of a weighted ensemble strategy for the Drude polarizable force field using the WESTPA software package in conjunction with the OpenMM simulation engine. We demonstrate its use for backbone conformational sampling in a model system (alanine dipeptide) and in studying functional sidechain rotations in an enzyme (Abl1 kinase). We further discuss possible consequences related to the inclusion of explicit electronic polarization in the model. This software implementation and validation should facilitate the use of WESTPA-Drude strategies throughout the simulation community.
Polysaccharide-based H₂S donors: Thiol-ene functionalization of amylopectin with H₂S-releasing N-thiocarboxyanhydrides
Chinn, Abigail F.; Williams, Noah R.; Miller, Kevin M.; Matson, John B. (Wiley, 2024-09-15)
Polymeric donors of gasotransmitters, gaseous signaling molecules such as hydrogen sulfide, nitric oxide, and carbon monoxide, hold potential for localized and extended delivery of these reactive gases. Examples of gasotransmitter donors based on polysaccharides are limited despite the availability and generally low toxicity of this broad class of polymers. In this work, we sought to create a polysaccharide H2S donor by covalently attaching N-thiocarboxyanhydrides (NTAs) to amylopectin, the major component of starch. To accomplish this, we added an allyl group to an NTA, which can spontaneously hydrolyze to release carbonyl sulfide and ultimately H2S via the ubiquitous enzyme carbonic anhydrase, and then coupled it to thiol-functionalized amylopectin of three different molecular weights (MWs) through thiol-ene "click" photochemistry. We also varied the degree of substitution (DS) of the NTA along the amylopectin backbone. H2S release studies on the six samples, termed amyl-NTAs, with variable MWs (three) and DS values (two), revealed that lower MW and higher DS led to faster release. Finally, dynamic light scattering experiments suggested that aggregation increased with MW, which may also have affected H2S release rates. Collectively, these studies present a new synthetic method to produce polysaccharide H2S donors for applications in the biomedical field.
Kinetic Characterization and Identification of Key Active Site Residues of the L-Aspartate N-Hydroxylase, CreE
Johnson, Sydney B.; Valentino, Hannah; Sobrado, Pablo (Wiley-V C H Verlag, 2024-07-15)
CreE is a flavin-dependent monooxygenase (FMO) that catalyzes three sequential nitrogen oxidation reactions of L-aspartate to produce nitrosuccinate, contributing to the biosynthesis of the antimicrobial and antiproliferative nautral product, cremeomycin. This compound contains a highly reactive diazo functional group for which the reaction of CreE is essential to its formation. Nitro and diazo functional groups can serve as potent electrophiles, important in some challenging nucleophilic addition reactions. Formation of these reactive groups positions CreE as a promising candidate for biomedical and synthetic applications. Here, we present the catalytic mechanism of CreE and the identification of active site residues critical to binding L-aspartate, aiding in future enzyme engineering efforts. Steady-state analysis demonstrated that CreE is very specific for NADPH over NADH and performs a highly coupled reaction with L-aspartate. Analysis of the rapid-reaction kinetics showed that flavin reduction is very fast, along with the formation of the oxygenating species, the C4a-hydroperoxyflavin. The slowest step observed was the dehydration of the flavin. Structural analysis and site-directed mutagenesis implicated T65, R291, and R440 in the binding L-aspartate. The data presented describes the catalytic mechanism and the active site architecture of this unique FMO.
Discovery of Potent, Orally Bioavailable Sphingosine-1-Phosphate Transporter (Spns2) Inhibitors
Foster, Daniel J.; Dunnavant, Kyle; Shrader, Christopher W.; LoPresti, Marion; Seay, Sarah; Kharel, Yugesh; Brown, Anne M.; Huang, Tao; Lynch, Kevin R.; Santos, Webster L. (American Chemical Society, 2024-07-02)
Targeting the S1P pathway has resulted in the development of S1P1 receptor modulators for the treatment of multiple sclerosis and ulcerative colitis. We hypothesize that targeting an upstream node of the S1P pathway may provide an improved adverse event profile. In this report, we performed a structure-activity relationship study focusing on the benzoxazole scaffold in SLB1122168, which lead to the discovery of 11i (SLF80821178) as a potent inhibitor of S1P release from HeLa cells (IC50: 51 +/- 3 nM). Administration of SLF80821178 to mice induced similar to 50% reduction in circulating lymphocyte counts, recapitulating the lymphopenia characteristic of Spns2 null animals. Molecular modeling studies suggest that SLF80821178 binds Spns2 in its occluded inward-facing state and forms hydrogen bonds with Asn112 and Ser211 and pi stacking with Phe234. Taken together, SLF80821178 can serve as a scaffold for future inhibitor development and represents a chemical tool to study the therapeutic implication of inhibiting Spns2.
Design, synthesis and biological evaluation of glucose metabolism inhibitors as anticancer agents
Cheng, Yao; Jones, John Patrick; Yu, Tsz Tin; Olzomer, Ellen M.; Su, Jacky; Katen, Alice; Black, David StC; Hart-Smith, Gene; Childress, Elizabeth S.; Wilkins, Marc R.; Mateos, Isabel A.; Santos, Webster L.; Hoehn, Kyle L.; Byrne, Frances L.; Kumar, Naresh (Academic Press-Elsevier, 2024-10-01)
Compared to normal cells, tumour cells exhibit an upregulation of glucose transporters and an increased rate of glycolytic activity. In previous research, we successfully identified a promising hit compound BH10 through a rigorous screening process, which demonstrates a potent capacity for inhibiting cancer cell proliferation by targeting glucose metabolism. In the current study, we identify Kelch-like ECH-associated protein 1 (Keap1) as a potential protein target of BH10 via avidin pull-down assays with biotinylated-BH10. Subsequently, we present a comprehensive analysis of a series of BH10 analogues characterized by the incorporation of a naphthoimidazole scaffold and the introduction of a triazole ring with diverse terminal functional groups. Notably, compound 4d has emerged as the most potent candidate, exhibiting better anti-cancer activities against HEC1A cancer cells with an IC50 of 2.60 mu M, an extended biological half-life, and an improved pharmacokinetic profile (compared to BH10) in mice.
Quantifying Induced Dipole Effects in Small Molecule Permeation in a Model Phospholipid Bilayer
Montgomery, Julia M.; Lemkul, Justin A. (American Chemical Society, 2024-07-22)
The cell membrane functions as a semipermeable barrier that governs the transport of materials into and out of cells. The bilayer features a distinct dielectric gradient due to the amphiphilic nature of its lipid components. This gradient influences various aspects of small molecule permeation and the folding and functioning of membrane proteins. Here, we employ polarizable molecular dynamics simulations to elucidate the impact of the electronic environment on the permeation process. We simulated eight distinct amino-acid side chain analogs within a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer using the Drude polarizable force field (FF). Our approach includes both unbiased and umbrella sampling simulations. By using a polarizable FF, we sought to investigate explicit dipole responses in relation to local electric fields along the membrane normal. We evaluate molecular dipole moments, which exhibit variation based on their localization within the membrane, and compare the outcomes with analogous simulations using the nonpolarizable CHARMM36 FF. This comparative analysis aims to discern characteristic differences in the free energy surfaces of permeation for the various amino-acid analogs. Our results provide the first systematic quantification of the impact of employing an explicitly polarizable FF in this context compared to the fixed-charge convention inherent to nonpolarizable FFs, which may not fully capture the influence of the membrane dielectric gradient.
Beneficial effects of MGL-3196 and BAM15 combination in a mouse model of fatty liver disease
Zhou, Mingyan; Li, Catherine; Byrne, Frances L.; Vancuylenburg, Calum S.; Olzomer, Ellen M.; Hargreaves, Adam; Wu, Lindsay E.; Shackel, Nicholas A.; Santos, Webster L.; Hoehn, Kyle L. (Wiley, 2024-10-01)
Background and Aim: Metabolic dysfunction-associated steatohepatitis (MASH) is a metabolic disorder with limited treatment options. The thyroid hormone receptor (THR)-beta agonist resmetirom/MGL-3196 (MGL) increases liver fat oxidation and has been approved for treating adult MASH. However, over 60% of patients receiving MGL treatment do not achieve MASH resolution. Therefore, we investigated the potential for combination therapy of MGL with the mitochondrial uncoupler BAM15 to improve fatty liver disease outcomes in the GAN mouse model of MASH. Methods: C57BL/6J male mice were fed GAN diet for 38 weeks before stratification and randomization to treatments including MGL, BAM15, MGL + BAM15, or no drug control for 8 weeks. Treatments were admixed in diet and mice were pair-fed to control for drug intake. Treatment effectiveness was assessed by body weight, body composition, energy expenditure, glucose tolerance, tissue lipid content, and histological analyses. Results: MGL + BAM15 treatment resulted in better efficacy versus GAN control mice than either monotherapy in the context of energy expenditure, liver fat loss, glucose control, and fatty liver disease activity score. Improvements in ALT, liver mass, and plasma cholesterol were primarily driven by MGL, while improvements in body fat were primarily driven by BAM15. No treatments altered liver fibrosis. Conclusions: MGL + BAM15 treatment had overall better efficacy to improve metabolic outcomes in mice fed GAN diet than either monotherapy alone. These data warrant further investigation into combination therapies of THR-beta agonists and mitochondrial uncouplers for the potential treatment of disorders related to fatty liver, obesity, and insulin resistance.
Deficiency of the sphingosine-1-phosphate (S1P) transporter Mfsd2b protects the heart against hypertension-induced cardiac remodeling by suppressing the L-type-Ca2+ channel
Duse, Dragos Andrei; Schroeder, Nathalie Hannelore; Srivastava, Tanu; Benkhoff, Marcel; Vogt, Jens; Nowak, Melissa Kim; Funk, Florian; Semleit, Nina; Wollnitzke, Philipp; Erkens, Ralf; Koetter, Sebastian; Meuth, Sven Guenther; Keul, Petra; Santos, Webster; Polzin, Amin; Kelm, Malte; Krueger, Martina; Schmitt, Joachim; Levkau, Bodo (Springer, 2024-10-01)
The erythrocyte S1P transporter Mfsd2b is also expressed in the heart. We hypothesized that S1P transport by Mfsd2b is involved in cardiac function. Hypertension-induced cardiac remodeling was induced by 4-weeks Angiotensin II (AngII) administration and assessed by echocardiography. Ca2+ transients and sarcomere shortening were examined in adult cardiomyocytes (ACM) from Mfsd2b(+/+) and Mfsd2b(-/-) mice. Tension and force development were measured in skinned cardiac fibers. Myocardial gene expression was determined by real-time PCR, Protein Phosphatase 2A (PP2A) by enzymatic assay, and S1P by LC/MS, respectively. Msfd2b was expressed in the murine and human heart, and its deficiency led to higher cardiac S1P. Mfsd2b(-/-) mice had regular basal cardiac function but were protected against AngII-induced deterioration of left-ventricular function as evidenced by similar to 30% better stroke volume and cardiac index, and preserved ejection fraction despite similar increases in blood pressure. Mfsd2b(-/-) ACM exhibited attenuated Ca2+ mobilization in response to isoprenaline whereas contractility was unchanged. Mfsd2b(-/-) ACM showed no changes in proteins responsible for Ca2+ homeostasis, and skinned cardiac fibers exhibited reduced passive tension generation with preserved contractility. Verapamil abolished the differences in Ca2+ mobilization between Mfsd2b(+/+) and Mfsd2b(-/-) ACM suggesting that S1P inhibits L-type-Ca2+ channels (LTCC). In agreement, intracellular S1P activated the inhibitory LTCC phosphatase PP2A in ACM and PP2A activity was increased in Mfsd2b(-/-) hearts. We suggest that myocardial S1P protects from hypertension-induced left-ventricular remodeling by inhibiting LTCC through PP2A activation. Pharmacologic inhibition of Mfsd2b may thus offer a novel approach to heart failure.
Ethyl cellulose-block-poly(benzyl glutamate) block copolymer compatibilizers for ethyl cellulose/poly(ethylene terephthalate) blends
Chinn, Abigail F.; Trindade Coutinho, Isabela; Kethireddy, Saipranavi Reddy; Williams, Noah R.; Knott, Kenneth M.; Moore, Robert B.; Matson, John B. (Royal Society Chemistry, 2024-08-27)
Blends of petroleum-based polymers with bio-sourced polymers are an alternative to polymers derived from non-renewable resources. However, polymer blends are usually immiscible, and a compatibilizer, often a block copolymer, is required to improve mixing. In this work, we synthesized a block copolymer of ethyl cellulose (ECel) and poly(benzyl glutamate), termed ECel-block-poly(BG), and we applied it as a compatibilizer for ECel/poly(ethylene terephthalate) (ECel/PET) blends. To synthesize this block copolymer, two ECel-NH2 macroinitiators were evaluated for ring-opening polymerization of benzyl glutamate-N-thiocarboxyanhydride (BG-NTA), one with the amine directly attached to the ECel reducing chain end, and the other with a short PEG linker between ECel and the amine initiator. The PEG-containing macroinitiator led to the synthesis of a block copolymer that was unimodal by size-exclusion chromatography (SEC) while the other initiator led to uncontrolled homopolymerization of BG-NTA, presumably due to steric hindrance near the primary amine. A series of solvent studies revealed that polymerization of BG-NTA in CH2Cl2 was the best system for obtaining the ECel-block-poly(BG) block copolymer, achieving 95% conversion based on H-1 NMR spectroscopy. The success of chain extension and molecular weight analysis were evaluated using SEC with multi-angle light scattering (SEC-MALS). Blends composed of 70% ECel and 30% PET with different weight percentages (wt%) of block copolymer compatibilizer were made via solvent casting from hexafluoroisopropanol. Phase contrast optical microscopy and small-angle laser light scattering were used to probe the effectiveness of the ECel-block-poly(BG) block copolymer as a compatibilizer (5-30 wt%) for the 70/30 ECel/PET blends. A decrease in average domain size from 15 +/- 4 mu m in the base blend (without compatibilizer) to 2 +/- 1 mu m in the blend containing 30 wt% ECel-block-poly(BG) indicated successful compatibilization of the blend.
Structural and Electronic Properties of Poly(ethylene terephthalate) (PET) from Polarizable Molecular Dynamics Simulations
Poleto, Marcelo D.; Lemkul, Justin A. (American Chemical Society, 2024-11-08)
The environmental and economic challenges posed by the widespread use and disposal of plastics, particularly poly(ethylene terephthalate) (PET), require innovative solutions to mitigate their impact. Such mitigation begins with understanding physical properties of the polymer that could enable new recycling technologies. Although molecular simulations have provided valuable insights into PET interactions with various PET hydrolases, current nonpolarizable force fields neglect the electronic polarization effects inherent to PET interactions. Here, we present parameters for PET polymer and its derivatives that are compatible with the Drude polarizable force field. Our parameter fitting protocol accurately reproduces electrostatic properties from quantum mechanical calculations. We then studied electronic properties of PET amorphous slabs and PET crystal units, revealing a crucial electronic polarization response of PET residues at the interface with water or vacuum, yielding insights into the modulation of electrostatic properties by solvent molecules. Finally, we showcase the interaction between a carbohydrate-binding protein and the PET crystal unit, revealing the role of electronic polarization in enhancing binding affinity. This study represents the first extension of the Drude polarizable force field to a synthetic polymer, offering a robust tool for exploring PET material properties and advancing the design of efficient (bio)technologies for addressing plastic pollution.
Design, Synthesis, and Biological Evaluation of [1,2,5]Oxadiazolo[3,4-b]pyridin-7-ol as Mitochondrial Uncouplers for the Treatment of Obesity and Metabolic Dysfunction-Associated Steatohepatitis
Foutz, Mary A.; Krinos, Emily L.; Beretta, Martina; Hargett, Stefan R.; Shrestha, Riya; Murray, Jacob H.; Duerre, Ethan; Salamoun, Joseph M.; Mccarter, Katrina; Shah, Divya P.; Hoehn, Kyle L.; Santos, Webster L. (American Chemical Society, 2024-11-30)
Mitochondrial uncouplers are small molecule protonophores that act to dissipate the proton motive force independent of adenosine triphosphate (ATP) synthase. Mitochondrial uncouplers such as BAM15 increase respiration and energy expenditure and have potential in treating a variety of metabolic diseases. In this study, we disclose the structure-activity relationship profile of 6-substituted [1,2,5]oxadiazolo[3,4-b]pyridin-7-ol derivatives of BAM15. Utilizing an oxygen consumption rate assay as a measure of increased cellular respiration, SHO1122147 (7m) displayed an EC50 of 3.6 mu M in L6 myoblasts. Pharmacokinetic studies indicated a half-life of 2 h, C max of 35 mu M, and no observed adverse effects at 1,000 mg kg-1 dose in mice. In a Gubra-Amylin (GAN) mouse model of MASH, SHO1122147 was efficacious in decreasing body weight and liver triglyceride levels at 200 mg kg-1 day-1 without changes in body temperature. These findings indicate the potential of utilizing novel [1,2,5]oxadiazolo[3,4-b]pyridin-7-ol mitochondrial uncouplers for treatment of fatty liver disease and obesity.
A Phage-Based Approach to Identify Antivirulence Inhibitors of Bacterial Type IV Pili
Shimozono, Tori M.; Vogelaar, Nancy J.; O'Hara, Megan T.; Yang, Zhaomin (Wiley, 2025-01-17)
The increasing threat of antibiotic resistance underscores the urgent need for innovative strategies to combat infectious diseases, including the development of antivirulants. Microbial pathogens rely on their virulence factors to initiate and sustain infections. Antivirulants are small molecules designed to target virulence factors, thereby attenuating the virulence of infectious microbes. The bacterial type IV pilus (T4P), an extracellular protein filament that depends on the T4P machinery (T4PM) for its biogenesis, dynamics and function, is a key virulence factor in many significant bacterial pathogens. While the T4PM presents a promising antivirulence target, the systematic identification of inhibitors for its multiple protein constituents remains a considerable challenge. Here we report a novel high-throughput screening (HTS) approach for discovering T4P inhibitors. It uses Pseudomonas aeruginosa, a high-priority pathogen, in combination with its T4P-targeting phage, φKMV. Screening of a library of 2168 compounds using an optimised protocol led to the identification of tuspetinib, based on its deterrence of the lysis of P. aeruginosa by φKMV. Our findings show that tuspetinib also inhibits two additional T4P-targeting phages, while having no effect on a phage that recognises lipopolysaccharides as its receptor. Additionally, tuspetinib impedes T4P-mediated motility in P. aeruginosa and Acinetobacter species without impacting growth or flagellar motility. This bacterium-phage pairing approach is applicable to a broad range of virulence factors that are required for phage infection, paving ways for the development of advanced chemotherapeutics against antibiotic-resistant infections.
Identification of small molecule inhibitors of the Chloracidobacterium thermophilum type IV pilus protein PilB by ensemble virtual screening
McDonald-Ramos, Jay S.; Hicklin, Ian K.; Yang, Zhaomin; Brown, Anne M. (Elsevier, 2024-08-16)
Antivirulence strategy has been explored as an alternative to traditional antibiotic development. The bacterial type IV pilus is a virulence factor involved in host invasion and colonization in many antibiotic resistant pathogens. The PilB ATPase hydrolyzes ATP to drive the assembly of the pilus filament from pilin subunits. We evaluated Chloracidobacterium thermophilum PilB (CtPilB) as a model for structure-based virtual screening by molecular docking and molecular dynamics (MD) simulations. A hexameric structure of CtPilB was generated through homology modeling based on an existing crystal structure of a PilB from Geobacter metallireducens. Four representative structures were obtained from molecular dynamics simulations to examine the conformational plasticity of PilB and improve docking analyses by ensemble docking. Structural analyses after 1 μs of simulation revealed conformational changes in individual PilB subunits are dependent on ligand presence. Further, ensemble virtual screening of a library of 4234 compounds retrieved from the ZINC15 database identified five promising PilB inhibitors. Molecular docking and binding analyses using the four representative structures from MD simulations revealed that top-ranked compounds interact with multiple Walker A residues, one Asp-box residue, and one arginine finger, indicating these are key residues in inhibitor binding within the ATP binding pocket. The use of multiple conformations in molecular screening can provide greater insight into compound flexibility within receptor sites and better inform future drug development for therapeutics targeting the type IV pilus assembly ATPase.
Balancing Group 1 Monoatomic Ion-Polar Compound Interactions in the Polarizable Drude Force Field: Application in Protein and Nucleic Acid Systems
Nan, Yiling; Baral, Prabin; Orr, Asuka A.; Michel, Haley M.; Lemkul, Justin A.; Mackerell, Alexander D. (American Chemical Society, 2024-12-03)
An accurate force field (FF) is the foundation of reliable results from molecular dynamics (MD) simulations. In our recently published work, we developed a protocol to generate atom pair-specific Lennard-Jones (known as NBFIX in CHARMM) and through-space Thole dipole screening (NBTHOLE) parameters in the context of the Drude polarizable FF based on readily accessible quantum mechanical (QM) data to fit condensed phase experimental thermodynamic benchmarks, including the osmotic pressure, diffusion coefficient, ionic conductivity, and solvation free energy, when available. In the present work, the developed protocol is applied to generate NBFIX and NBTHOLE parameters for interactions between monatomic ions (specifically Li+, Na+, K+, Rb+, Cs+, and Cl-) and common functional groups found in proteins and nucleic acids. The parameters generated for each ion-functional group pair were then applied to the corresponding functional groups within proteins or nucleic acids followed by MD simulations to analyze the distribution of ions around these biomolecules. The modified FF successfully addresses the issue of overbinding observed in a previous iteration of the Drude FF. Quantitatively, the model accurately reproduces the effective charge of proteins and demonstrates a level of charge neutralization for a double-helix B-DNA in good agreement with the counterion condensation theory. Additionally, simulations involving ion competition correlate well with experimental results, following the trend Li+ > Na+ ≈ K+ > Rb+. These results validate the refined model for group 1 ion-biomolecule interactions that will facilitate the application of the polarizable Drude FF in systems in which group 1 ions play an important role.

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