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- A Phage-Based Approach to Identify Antivirulence Inhibitors of Bacterial Type IV PiliShimozono, 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 screeningMcDonald-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 SystemsNan, 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 GROMACSLemkul, 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-peptideDavidson, 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 junctionMichel, 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 communitiesGoldberg, 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 MonooxygenaseJohnson, 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-QuadruplexesPoleto, 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.
- Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence CompoundsDye, Keane J.; Vogelaar, Nancy J.; O'Hara, Megan; Sobrado, Pablo; Santos, Webster; Carlier, Paul R.; Yang, Zhaomin (American Society for Microbiology, 2022-12)Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo. We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson's disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics.IMPORTANCE Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections.
- Targeting negative energy balance with calorie restriction and mitochondrial uncoupling in db/db miceChen, Sing -Young; Beretta, Martina; Olzomer, Ellen M.; Shah, Divya P.; Wong, Derek Y. H.; Alexopoulos, Stephanie J.; Aleksovska, Isabella; Salamoun, Joseph M.; Garcia, Christopher J.; Cochran, Blake J.; Rye, Kerry-Anne; Smith, Greg C.; Byrne, Frances L.; Morris, Margaret J.; Santos, Webster L.; Cantley, James; Hoehn, Kyle L. (Elsevier, 2023-01)Objective: Calorie restriction is a first-line treatment for overweight individuals with metabolic impairments. However, few patients can adhere to long-term calorie restriction. An alternative approach to calorie restriction that also causes negative energy balance is mitochondrial uncoupling, which decreases the amount of energy that can be extracted from food. Herein we compare the metabolic effects of calorie restriction with the mitochondrial uncoupler BAM15 in the db/db mouse model of severe hyperglycemia, obesity, hypertriglyceridemia, and fatty liver. Methods: Male db/db mice were treated with w50% calorie restriction, BAM15 at two doses of 0.1% and 0.2% (w/w) admixed in diet, or 0.2% BAM15 with time-restricted feeding from 5 weeks of age. Mice were metabolically phenotyped over 4 weeks with assessment of key readouts including body weight, glucose tolerance, and liver steatosis. At termination, liver tissues were analysed by metabolomics and qPCR. Results: Calorie restriction and high-dose 0.2% BAM15 decreased body weight to a similar extent, but mice treated with BAM15 had far better improvement in glucose control. High-dose BAM15 treatment completely normalized fasting glucose and glucose tolerance to levels similar to lean db/+ control mice. Low-dose 0.1% BAM15 did not affect body mass but partially improved glucose tolerance to a similar degree as 50% calorie restriction. Both calorie restriction and high-dose BAM15 significantly improved hyperglucagonemia and liver and serum triglyceride levels. Combining high-dose BAM15 with time-restricted feeding to match the time that calorie restricted mice were fed resulted in the best metabolic phenotype most similar to lean db/+ controls. BAM15-mediated improvements in glucose control were associated with decreased glucagon levels and decreased expression of enzymes involved in hepatic gluconeogenesis. Conclusions: BAM15 and calorie restriction treatments improved most metabolic disease phenotypes in db/db mice. However, mice fed BAM15 had superior effects on glucose control compared to the calorie restricted group that consumed half as much food. Submaximal dosing with BAM15 demonstrated that its beneficial effects on glucose control are independent of weight loss. These data highlight the potential for mitochondrial uncoupler pharmacotherapies in the treatment of metabolic disease. (c) 2023 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
- Sphingosine Kinase 2 Inhibitors: Rigid Aliphatic Tail Derivatives Deliver Potent and Selective AnaloguesPashikanti, Srinath; Foster, Daniel J.; Kharel, Yugesh; Brown, Anne M.; Bevan, David R.; Lynch, Kevin R.; Santos, Webster L. (American Chemical Society, 2022-10-19)Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that interacts with five native G-protein coupled receptors (S1P1-5) to regulate cell growth, survival, and proliferation. S1P has been implicated in a variety of pathologies including cancer, kidney fibrosis, and multiple sclerosis. As key mediators in the synthesis of S1P, sphingosine kinase (SphK) isoforms 1 and 2 have attracted attention as viable targets for pharmacologic intervention. In this report, we describe the design, synthesis, and biological evaluation of sphingosine kinase 2 (SphK2) inhibitors with a focus on systematically introducing rigid structures in the aliphatic lipid tail present in existing SphK2 inhibitors. Experimental as well as molecular modeling studies suggest that conformationally restricted "lipophilic tail" analogues bearing a bulky terminal moiety or an internal phenyl ring are useful to complement the "J"-shaped sphingosine binding pocket of SphK2. We identified 14c (SLP9101555) as a potent SphK2 inhibitor (Ki= 90 nM) with 200-fold selectivity over SphK1. Molecular docking studies indicated key interactions: the cyclohexyl ring binding in the cleft deep in the pocket, a trifluoromethyl group fitting in a small side cavity, and a hydrogen bond between the guanidino group and Asp308 (amino acid numbering refers to human SphK2 (isoform c) orthologue). In vitro studies using U937 human histiocytic lymphoma cells showed marked decreases in extracellular S1P levels in response to our SphK2 inhibitors. Administration of 14c (dose: 5 mg/kg) to mice resulted in a sustained increase of circulating S1P levels, suggesting target engagement.
- N-thiocarboxyanhydrides, amino acid-derived enzyme-activated H2S donors, enhance sperm mitochondrial activity in presence and absence of oxidative stressPintus, Eliana; Chinn, Abigail F.; Kadlec, Martin; García-Vázquez, Francisco A.; Novy, Pavel; Matson, John B.; Ros-Santaella, José L. (2023-02-16)Background Hydrogen sulfide (H2S) donors are crucial tools not only for understanding the role of H2S in cellular function but also as promising therapeutic agents for oxidative stress-related diseases. This study aimed to explore the effect of amino acid-derived N-thiocarboxyanhydrides (NTAs), which release physiological H2S levels in the presence of carbonic anhydrase, on porcine sperm function during short-term incubation with and without induced oxidative stress. For this purpose, we employed two H2S-releasing NTAs with release half-lives (t1/2) in the range of hours that derived from the amino acids glycine (Gly-NTA) or leucine (Leu-NTA). Because carbonic anhydrase is crucial for H2S release from NTAs, we first measured the activity of this enzyme in the porcine ejaculate. Then, we tested the effect of Gly- and Leu-NTAs at 10 and 1 nM on sperm mitochondrial activity, plasma membrane integrity, acrosomal status, motility, motile subpopulations, and redox balance during short-term incubation at 38 °C with and without a reactive oxygen species (ROS)-generating system. Results Our results show that carbonic anhydrase is found both in spermatozoa and seminal plasma, with activity notably higher in the latter. Both Gly- and Leu-NTAs did not exert any noxious effects, but they enhanced sperm mitochondrial activity in the presence and absence of oxidative stress. Moreover, NTAs (except for Leu-NTA 10 nM) tended to preserve the sperm redox balance against the injuries provoked by oxidative stress, which provide further support to the antioxidant effect of H2S on sperm function. Both compounds also increased progressive motility over short-term incubation, which may translate into prolonged sperm survival. Conclusions The presence of carbonic anhydrase activity in mammalian spermatozoa makes NTAs promising molecules to investigate the role of H2S in sperm biology. For the first time, beneficial effects of NTAs on mitochondrial activity have been found in mammalian cells in the presence and absence of oxidative stress. NTAs are interesting compounds to investigate the role of H2S in sperm mitochondria-dependent events and to develop H2S-related therapeutic protocols against oxidative stress in assisted reproductive technologies.
- An olive-derived elenolic acid stimulates hormone release from L-cells and exerts potent beneficial metabolic effects in obese diabetic miceWang, Yao; Wu, Yajun; Wang, Aiping; Wang, Aihua; Alkhalidy, Hana; Helm, Richard; Zhang, Shijun; Ma, Hongguang; Zhang, Yan; Gilbert, Elizabeth R.; Xu, Bin; Liu, Dongmin (Frontiers, 2022-11-01)Insulin resistance and progressive decline in functional β-cell mass are two key factors for developing type 2 diabetes (T2D), which is largely driven by overweight and obesity, a significant obstacle for effective metabolic control in many patients with T2D. Thus, agents that simultaneously ameliorate obesity and act on multiple pathophysiological components could be more effective for treating T2D. Here, we report that elenolic acid (EA), a phytochemical, is such a dual-action agent. we show that EA dose-dependently stimulates GLP-1 secretion in mouse clonal L-cells and isolated mouse ileum crypts. In addition, EA induces L-cells to secrete peptide YY (PYY). EA induces a rapid increase in intracellular [Ca2+]i and the production of inositol trisphosphate in L-cells, indicating that EA activates phospholipase C (PLC)-mediated signaling. Consistently, inhibition of (PLC) or Gαq ablates EA-stimulated increase of [Ca2+]i and GLP-1 secretion. In vivo, a single dose of EA acutely stimulates GLP-1 and PYY secretion in mice, accompanied with an improved glucose tolerance and insulin levels. Oral administration of EA at a dose of 50 mg/kg/day for 2 weeks normalized the fasting blood glucose and restored glucose tolerance in high-fat diet-induced obese (DIO) mice to levels that were comparable to chow-fed mice. In addition, EA suppresses appetite, reduces food intake, promotes weight loss, and reverses perturbated metabolic variables in obese mice. These results suggest that EA could be a dual-action agent as an alternative or adjuvant treatment for both T2D and obesity.
- Kinetic and Structural Characterization of a Flavin-Dependent Putrescine N-Hydroxylase from Acinetobacter baumanniiLyons, Noah S.; Bogner, Alexandra N.; Tanner, John J.; Sobrado, Pablo (American Chemical Society, 2022-11-15)Acinetobacter baumannii is a Gram-negative opportunistic pathogen that causes nosocomial infections, especially among immunocompromised individuals. The rise of multidrug resistant strains of A. baumannii has limited the use of standard antibiotics, highlighting a need for new drugs that exploit novel mechanisms of pathogenicity. Disrupting iron acquisition by inhibiting the biosynthesis of iron-chelating molecules (siderophores) secreted by the pathogen is a potential strategy for developing new antibiotics. Here we investigated FbsI, an N-hydroxylating monooxygenase involved in the biosynthesis of fimsbactin A, the major siderophore produced by A. baumannii. FbsI was characterized using steady-state and transient-state kinetics, spectroscopy, X-ray crystallography, and small-angle X-ray scattering. FbsI was found to catalyze the N-hydroxylation of the aliphatic diamines putrescine and cadaverine. Maximum coupling of the reductive and oxidative half-reactions occurs with putrescine, suggesting it is the preferred (in vivo) substrate. FbsI uses both NADPH and NADH as the reducing cofactor with a slight preference for NADPH. The crystal structure of FbsI complexed with NADP+was determined at 2.2 Å resolution. The structure exhibits the protein fold characteristic of Class B flavin-dependent monooxygenases. FbsI is most similar in 3D structure to the cadaverine N-hydroxylases DesB and DfoA. Small-angle X-ray scattering shows that FbsI forms a tetramer in solution like the N-hydroxylating monooxygenases of the SidA/IucD/PvdA family. A model of putrescine docked into the active site provides insight into substrate recognition. A mechanism for the catalytic cycle is proposed where dehydration of the C4a-hydroxyflavin intermediate is partially rate-limiting, and the hydroxylated putrescine product is released before NADP+
- TUPÃ: Electric field analyses for molecular simulationsPolêto, Marcelo D.; Lemkul, Justin A. (Wiley, 2022-04-22)We introduce TUPÃ, a Python-based algorithm to calculate and analyze electric fields in molecular simulations. To demonstrate the features in TUPÃ, we present three test cases in which the orientation and magnitude of the electric field exerted by biomolecules help explain biological phenomena or observed kinetics. As part of TUPÃ, we also provide a PyMOL plugin to help researchers visualize how electric fields are organized within the simulation system. The code is freely available and can be obtained at https://mdpoleto.github.io/tupa/.
- Electronic Polarization at the Interface between the p53 Transactivation Domain and Two Binding PartnersCorrigan, Alexsandra N.; Lemkul, Justin A. (American Chemical Society, 2022-07-07)Intrinsically disordered proteins (IDPs) are an abundant class of highly charged proteins that participate in numerous crucial biological processes, often in regulatory roles. IDPs do not have one major free energy minimum with a dominant structure, instead existing as conformational ensembles of multiple semistable conformations. p53 is a prototypical protein with disordered regions and binds to many structurally diverse partners, making it a useful model for exploring the role of electrostatic interactions at IDP binding interfaces. In this study, we used the Drude-2019 force field to simulate the p53 transactivation domain with two protein partners to probe the role of electrostatic interactions in IDP protein-protein interactions. We found that the Drude-2019 polarizable force field reasonably reproduced experimental chemical shifts of the p53 transactivation domain (TAD) in one complex for which these data are available. We also found that the proteins in these complexes displayed dipole response at specific residues of each protein and that residues primarily involved in binding showed a large percent change in dipole moment between the unbound and complexed states. Probing the role of electrostatic interactions in IDP binding can allow us greater fundamental understanding of these interactions and may help with targeting p53 or its partners for drug design.
- Effects of Familial Alzheimer's Disease Mutations on the Folding Free Energy and Dipole-Dipole Interactions of the Amyloid β-PeptideDavidson, Darcy S.; Kraus, Joshua A.; Montgomery, Julia M.; Lemkul, Justin A. (American Chemical Society, 2022-10-06)Familial Alzheimer's disease (FAD) mutations of the amyloid β-peptide (Aβ) are known to lead to early onset and more aggressive Alzheimer's disease. FAD mutations such as "Iowa" (D23N), "Arctic" (E22G), "Italian" (E22K), and "Dutch" (E22Q) have been shown to accelerate Aβ aggregation relative to the wild-type (WT). The mechanism by which these mutations facilitate increased aggregation is unknown, but each mutation results in a change in the net charge of the peptide. Previous studies have used nonpolarizable force fields to study Aβ, providing some insight into how this protein unfolds. However, nonpolarizable force fields have fixed charges that lack the ability to redistribute in response to changes in local electric fields. Here, we performed polarizable molecular dynamics simulations on the full-length Aβ42of WT and FAD mutations and calculated folding free energies of the Aβ15-27fragment via umbrella sampling. By studying both the full-length Aβ42and a fragment containing mutations and the central hydrophobic cluster (residues 17-21), we were able to systematically study how these FAD mutations impact secondary and tertiary structure and the thermodynamics of folding. Electrostatic interactions, including those between permanent and induced dipoles, affected side-chain properties, salt bridges, and solvent interactions. The FAD mutations resulted in shifts in the electronic structure and solvent accessibility at the central hydrophobic cluster and the hydrophobic C-terminal region. Using umbrella sampling, we found that the folding of the WT and E22 mutants is enthalpically driven, whereas the D23N mutant is entropically driven, arising from a different unfolding pathway and peptide-bond dipole response. Together, the unbiased, full-length, and umbrella sampling simulations of fragments reveal that the FAD mutations perturb nearby residues and others in hydrophobic regions to potentially alter solubility. These results highlight the role electronic polarizability plays in amyloid misfolding and the role of heterogeneous microenvironments that arise as conformational change takes place.
- Ion-Dependent Conformational Plasticity of Telomeric G-Hairpins and G-QuadruplexesSalsbury, Alexa M.; Michel, Haley M.; Lemkul, Justin A. (American Chemical Society, 2022-07-12)Telomeric DNA is guanine-rich and can adopt structures such as G-quadruplexes (GQs) and G-hairpins. Telomeric GQs influence genome stability and telomerase activity, making understanding of enzyme-GQ interactions and dynamics important for potential drug design. GQs have a characteristic tetrad core, which is connected by loop regions. Within this architecture are G-hairpins, fold back motifs that are thought to represent the first intermediate in GQ folding. To better understand the relationship between G-hairpin motifs and GQs, we performed polarizable simulations of a two-tetrad telomeric GQ and an isolated SC11 telomeric G-hairpin. The telomeric GQ contains a G-triad, which functions as part of the tetrad core or linker regions, depending on local conformational change. This triad and another motif below the tetrad core frequently bound ions and may represent druggable sites. Further, we observed the unbiased formation of a G-triad and a G-tetrad in simulations of the SC11 G-hairpin and found that cations can be partially hydrated while facilitating the formation of these motifs. Finally, we demonstrated that K+ ions form specific interactions with guanine bases, while Na+ ions interact nonspecifically with bases in the structure. Together, these simulations provide new insights into the influence of ions on GQs, G-hairpins, and G-triad motifs.
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