Scholarly Works, Virginia Tech Center for Drug Discovery

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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.
Introductory Tutorials for Simulating Protein Dynamics with GROMACS
Lemkul, Justin A. (American Chemical Society, 2024-09-21)
Atomistic molecular dynamics (MD) simulations have become an indispensable tool for investigating the structure, dynamics, and energetics of biomolecules. Continual optimization of software algorithms and hardware has enabled investigators to access biologically relevant time scales in feasible amounts of computing time. Given the widespread use and utility of MD simulations, there is considerable interest in learning essential skills in performing them. Here, we present a set of introductory tutorials for performing MD simulations of proteins in the popular, open-source GROMACS package. Three exercises are detailed, including simulating a single protein, setting up a protein complex, and performing umbrella sampling simulations to model the unfolding of a short polypeptide. Essential features and input settings are illustrated throughout. The purpose of these tutorials is to provide new users with a general understanding of foundational workflows, from which they can design their own simulations.
Pyroglutamylation modulates electronic properties and the conformational ensemble of the amyloid β-peptide
Davidson, Darcy S.; Lemkul, Justin A. (Wiley, 2024-03-04)
Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by the formation of extracellular amyloid-β (Aβ) plaques. The underlying cause of AD is unknown, however, post-translational modifications (PTMs) of Aβ have been found in AD patients and are thought to play a role in protein aggregation. One such PTM is pyroglutamylation, which can occur at two sites in Aβ, Glu3 and Glu11. This modification of Aβ involves the truncation and charge-neutralization of N-terminal glutamate, causing Aβ to become more hydrophobic and prone to aggregation. The molecular mechanism by which the introduction of pyroglutamate (pE) promotes aggregation has not been determined. To gain a greater understanding of the role that charge neutralization and truncation of the N-terminus plays on Aβ conformational sampling, we used the Drude polarizable force field (FF) to perform molecular dynamics simulations on AβpE3–42 and AβpE11–42 and comparing their properties to previous simulations of Aβ1–42. The Drude polarizable FF allows for a more accurate representation of electrostatic interactions, therefore providing novel insights into the role that charge plays in protein dynamics. Here, we report the parametrization of pE in the Drude polarizable FF and the effect of pyroglutamylation on Aβ. We found that AβpE3–42 and AβpE11–42 alter the permanent and induced dipoles of the peptide. Specifically, we found that AβpE3–42 and AβpE11–42 have modification-specific backbone and sidechain polarization response and perturbed solvation properties that shift the Aβ conformational ensemble.
Base pair dynamics, electrostatics, and thermodynamics at the LTR-III quadruplex:duplex junction
Michel, Haley M.; Lemkul, Justin A. (Cell Press, 2024-04-04)
G-quadruplexes (GQs) play key regulatory roles within the human genome and have also been identified to play similar roles in other eukaryotes, bacteria, archaea, and viruses. Human immunodeficiency virus 1, the etiological agent of acquired immunodeficiency syndrome, can form two GQs in its long terminal repeat (LTR) promoter region, each of which act to regulate viral gene expression in opposing manners. The major LTR GQ, called LTR-III, is a distinct hybrid GQ containing a 12-nucleotide duplex loop attached to the quadruplex motif. The resulting quadruplex:duplex junction (QDJ) has been hypothesized to serve as a selective drug targeting site. To better understand the dynamics of this QDJ, we performed conventional and enhanced-sampling molecular dynamics simulations using the Drude-2017 force field. We observed unbiased and reversible formation of additional base pairs in the QDJ, between Ade4:Thy14 and Gua3:Thy14. Both base pairs were electrostatically favored, but geometric constraints within the junction may drive the formation of, and preference for, the Ade4:Thy14 base pair. Finally, we demonstrated that the base pairs are separated only by small energy barriers that may enable transitions between both base-paired states. Together, these simulations provide new insights into the dynamics, electrostatics, and thermodynamics of the LTR-III QDJ.
Widespread exposure to SARS-CoV-2 in wildlife communities
Goldberg, Amanda R.; Langwig, Kate E.; Brown, Katherine L.; Marano, Jeffrey M.; Rai, Pallavi; King, Kelsie M.; Sharp, Amanda K.; Ceci, Alessandro; Kailing, Christopher D.; Kailing, Macy J.; Briggs, Russell; Urbano, Matthew G.; Roby, Clinton; Brown, Anne M.; Weger-Lucarelli, James; Finkielstein, Carla V.; Hoyt, Joseph R. (Springer, 2024-07-29)
Pervasive SARS-CoV-2 infections in humans have led to multiple transmission events to animals. While SARS-CoV-2 has a potential broad wildlife host range, most documented infections have been in captive animals and a single wildlife species, the white-tailed deer. The full extent of SARS-CoV-2 exposure among wildlife communities and the factors that influence wildlife transmission risk remain unknown. We sampled 23 species of wildlife for SARS-CoV-2 and examined the effects of urbanization and human use on seropositivity. Here, we document positive detections of SARS-CoV-2 RNA in six species, including the deer mouse, Virginia opossum, raccoon, groundhog, Eastern cottontail, and Eastern red bat between May 2022–September 2023 across Virginia and Washington, D.C., USA. In addition, we found that sites with high human activity had three times higher seroprevalence than low human-use areas. We obtained SARS-CoV-2 genomic sequences from nine individuals of six species which were assigned to seven Pango lineages of the Omicron variant. The close match to variants circulating in humans at the time suggests at least seven recent human-to-animal transmission events. Our data support that exposure to SARS-CoV-2 has been widespread in wildlife communities and suggests that areas with high human activity may serve as points of contact for cross-species transmission.
Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase
Johnson, Sydney B.; Li, Hao; Valentino, Hannah; Sobrado, Pablo (American Chemical Society, 2024-05-23)
OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a “cautious” flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme−substrate and enzyme− intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.
Differences in Conformational Sampling and Intrinsic Electric Fields Drive Ion Binding in Telomeric and TERRA G-Quadruplexes
Poleto, Marcelo D.; Lemkul, Justin A. (American Chemical Society, 2023-10-17)
The formation of G-quadruplexes (GQs) occurs in guanine-rich sequences of DNA and RNA, producing highly stable and structurally diverse noncanonical nucleic acid structures. GQs play crucial roles in regulating transcription, translation, and replication and maintaining the genome, among others; thus, changes to their structures can lead to diseases such as cancer. Previous studies using polarizable molecular dynamics simulations have shown differences in ion binding properties between telomeric and telomeric repeat-containing RNA GQs despite architectural similarities. Here, we used volume-based metadynamics and repulsive potential simulations in conjunction with polarizable force fields to quantify the impact of ion binding on the GQ dynamics and ion binding free energies. Furthermore, we describe how GQs exert electric fields on their surroundings to link dynamics with variations in the electronic structure. Our findings provide new insights into the energetic, physical, and conformational properties of GQs and expose subtle but important differences between DNA and RNA GQs with the same fold.
Enzyme-Triggered Chemodynamic Therapy via a Peptide-H2S Donor Conjugate with Complexed Fe2+
Zhu, Yumeng; Archer, William R.; Morales, Katlyn F.; Schulz, Michael D.; Wang, Yin; Matson, John B. (Wiley-V C H Verlag, 2023-04)
Inducing high levels of reactive oxygen species (ROS) inside tumor cells is a cancer therapy method termed chemodynamic therapy (CDT). Relying on delivery of Fenton reaction promoters such as Fe2+, CDT takes advantage of overproduced ROS in the tumor microenvironment. We developed a peptide-H2S donor conjugate, complexed with Fe2+, termed AAN-PTC-Fe2+. The AAN tripeptide was specifically cleaved by legumain, an enzyme overexpressed in glioma cells, to release carbonyl sulfide (COS). Hydrolysis of COS by carbonic anhydrase formed H2S, an inhibitor of catalase, an enzyme that detoxifies H2O2. Fe2+ and H2S together increased intracellular ROS levels and decreased viability in C6 glioma cells compared with controls lacking either Fe2+, the AAN sequence, or the ability to generate H2S. AAN-PTC-Fe2+ performed better than temezolimide while exhibiting no cytotoxicity toward H9C2 cardiomyocytes. This study provides an H2S-amplified, enzyme-responsive platform for synergistic cancer treatment.

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