Scholarly Works, Virginia Tech Center for Drug Discovery

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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.
Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence Compounds
Dye, 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 mice
Chen, 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 Analogues
Pashikanti, 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 stress
Pintus, 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 mice
Wang, 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 baumannii
Lyons, 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 simulations
Polêto, 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 Partners
Corrigan, 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-Peptide
Davidson, 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-Quadruplexes
Salsbury, 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.
Identification of the Target for a Transition Metal-alpha-Amino Acid Complex Antibiotic Against Mycobacterium smegmatis
Karpin, George W.; Merola, Joseph S.; Falkinham, Joseph O. III (2021-06-25)
Spontaneous mutants of Mycobacterium smegmatis strain mc(2)155 resistant to 1-PG (iridium-L-phenylglycine complex), an antimycobacterial antibiotic, were isolated. Based on the discovery that some 1-PG-resistant mutants (1-PG(R)) were also resistant to high concentrations of clarithromycin (>= 250 mu g/ml), but no other anti-mycobacterial antibiotics, the 23S rRNA region spanning the peptidyl transferase domain was sequenced and mutations shown to be localized in the peptidyl transferase domain of the 23S rRNA gene. Measurements showed that 1-PG bound to ribosomes isolated from the 1-PG-sensitive parental strain, but the ribosome binding values for the 1-PG(R) mutant reduced.
Integration of experimental data and use of automated fitting methods in developing protein force fields
Polêto, Marcelo D.; Lemkul, Justin A. (Springer Nature, 2022-03-18)
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.
Cation competition and recruitment around the c-kit1 G-quadruplex using polarizable simulations
Salsbury, Alexa M.; Lemkul, Justin A. (2021-06-01)
Nucleic acid-ion interactions are fundamentally important to the physical, energetic, and conformational properties of DNA and RNA. These interactions help fold and stabilize highly ordered secondary and tertiary structures, such as G-quadruplexes (GQs), which are functionally relevant in telomeres, replication initiation sites, and promoter sequences. The c-kit protooncogene encodes for a receptor tyrosine kinase and is linked to gastrointestinal stromal tumors, mast cell disease, and leukemia. This gene contains three unique GQ-forming sequences that have proposed antagonistic effects on gene expression. The dominant GQ, denoted c-kit1, has been shown to decrease expression of c-kit transcripts, making the c-kit1GQa promising drug target. Toward disease intervention, more information is needed regarding its conformational dynamics and ion binding properties. Therefore, we performed molecular dynamics simulations of the c-kit1 GQ with K+, Na+, Li+, and mixed salt solutions using the Drude-2017 polarizable force field. We evaluated GQ structure, ion sampling, core energetics, ion dehydration and binding, and ion competition and found that each analysis supported the known GQ-ion specificity trend (K+ > Na+ > Li+). We also found that K+ ions coordinate in the tetrad core antiprismatically, whereas Na+ and Li+ align coplanar to guanine tetrads, partially because of their attraction to surrounding water. Further, we showed that K+ occupancy is higher around the c-kit1 GQ and its nucleobases than Na+ and Li+, which tend to interact with backbone and sugar moieties. Finally, we showed that K+ binding to the c-kit1GQ is faster and more frequent than Na+ and Li+. Such descriptions of GQ-ion dynamics suggest the rate of dehydration as the dominant factor for preference of K+ by DNA GQs and provide insight into noncanonical nucleic acids for which little experimental data exist.
The Pro-Inflammatory Chemokines CXCL9, CXCL10 and CXCL11 Are Upregulated Following SARS-CoV-2 Infection in an AKT-Dependent Manner
Callahan, Victoria; Hawks, Seth A.; Crawford, Matthew A.; Lehman, Caitlin W.; Morrison, Holly A.; Ivester, Hannah M.; Akhrymuk, Ivan V.; Boghdeh, Niloufar; Flor, Rafaela; Finkielstein, Carla V.; Allen, Irving C.; Weger-Lucarelli, James; Duggal, Nisha K.; Hughes, Molly A.; Kehn-Hall, Kylene (MDPI, 2021-06-03)
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a ‘cytokine storm.’ In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.
High-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilB
Dye, Keane J.; Vogelaar, Nancy J.; Sobrado, Pablo; Yang, Zhaomin (2021-03)
The bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitro. Chloracidobacterium thermophilum PilB (CtPilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized CtPilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro. Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable in seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems. IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.
Safety of drug use in patients with a primary mitochondrial disease: An international Delphi-based consensus
De Vries, Maaike C.; Brown, David A.; Allen, Mitchell E.; Bindoff, Laurence; Gorman, Grainne S.; Karaa, Amel; Keshavan, Nandaki; Lamperti, Costanza; McFarland, Robert; Ng, Yi Shiau; O'Callaghan, Mar; Pitceathly, Robert D. S.; Rahman, Shamima; Russel, Frans G. M.; Varhaug, Kristin N.; Schirris, Tom J. J.; Mancuso, Michelangelo (2020-07)
Clinical guidance is often sought when prescribing drugs for patients with primary mitochondrial disease. Theoretical considerations concerning drug safety in patients with mitochondrial disease may lead to unnecessary withholding of a drug in a situation of clinical need. The aim of this study was to develop consensus on safe medication use in patients with a primary mitochondrial disease. A panel of 16 experts in mitochondrial medicine, pharmacology, and basic science from six different countries was established. A modified Delphi technique was used to allow the panellists to consider draft recommendations anonymously in two Delphi rounds with predetermined levels of agreement. This process was supported by a review of the available literature and a consensus conference that included the panellists and representatives of patient advocacy groups. A high level of consensus was reached regarding the safety of all 46 reviewed drugs, with the knowledge that the risk of adverse events is influenced both by individual patient risk factors and choice of drug or drug class. This paper details the consensus guidelines of an expert panel and provides an important update of previously established guidelines in safe medication use in patients with primary mitochondrial disease. Specific drugs, drug groups, and clinical or genetic conditions are described separately as they require special attention. It is important to emphasise that consensus-based information is useful to provide guidance, but that decisions related to drug prescribing should always be tailored to the specific needs and risks of each individual patient. We aim to present what is current knowledge and plan to update this regularly both to include new drugs and to review those currently included.
The cardiolipin-binding peptide elamipretide mitigates fragmentation of cristae networks following cardiac ischemia reperfusion in rats
Allen, Mitchell E.; Pennington, Edward Ross; Perry, Justin B.; Dadoo, Sahil; Makrecka-Kuka, Marina; Dambrova, Maija; Moukdar, Fatiha; Patel, Hetal D.; Han, Xianlin; Kidd, Grahame K.; Benson, Emily K.; Raisch, Tristan B.; Poelzing, Steven; Brown, David A.; Shaikh, Saame Raza (2020-07-17)
Allen and Pennington et al. show that the cardiolipin-binding peptide elamipretide mitigates disease-induced fragmentation of cristae networks following cardiac ischemia reperfusion in rats. This study suggests that elamipretide targets mitochondrial membranes to sustain cristae networks, improving their bioenergetic function. Mitochondrial dysfunction contributes to cardiac pathologies. Barriers to new therapies include an incomplete understanding of underlying molecular culprits and a lack of effective mitochondria-targeted medicines. Here, we test the hypothesis that the cardiolipin-binding peptide elamipretide, a clinical-stage compound under investigation for diseases of mitochondrial dysfunction, mitigates impairments in mitochondrial structure-function observed after rat cardiac ischemia-reperfusion. Respirometry with permeabilized ventricular fibers indicates that ischemia-reperfusion induced decrements in the activity of complexes I, II, and IV are alleviated with elamipretide. Serial block face scanning electron microscopy used to create 3D reconstructions of cristae ultrastructure reveals that disease-induced fragmentation of cristae networks are improved with elamipretide. Mass spectrometry shows elamipretide did not protect against the reduction of cardiolipin concentration after ischemia-reperfusion. Finally, elamipretide improves biophysical properties of biomimetic membranes by aggregating cardiolipin. The data suggest mitochondrial structure-function are interdependent and demonstrate elamipretide targets mitochondrial membranes to sustain cristae networks and improve bioenergetic function.
Structural, in silico, and functional analysis of a Disabled-2-derived peptide for recognition of sulfatides
Song, Wei; Gottschalk, Carter J.; Tang, Tuo-Xian; Biscardi, Andrew; Ellena, Jeffrey F.; Finkielstein, Carla V.; Brown, Anne M.; Capelluto, Daniel G. S. (2020-08-11)
Disabled-2 (Dab2) is an adaptor protein that regulates the extent of platelet aggregation by two mechanisms. In the first mechanism, Dab2 intracellularly downregulates the integrin alpha (IIb)beta (3) receptor, converting it to a low affinity state for adhesion and aggregation processes. In the second mechanism, Dab2 is released extracellularly and interacts with the pro-aggregatory mediators, the integrin alpha (IIb)beta (3) receptor and sulfatides, blocking their association to fibrinogen and P-selectin, respectively. Our previous research indicated that a 35-amino acid region within Dab2, which we refer to as the sulfatide-binding peptide (SBP), contains two potential sulfatide-binding motifs represented by two consecutive polybasic regions. Using molecular docking, nuclear magnetic resonance, lipid-binding assays, and surface plasmon resonance, this work identifies the critical Dab2 residues within SBP that are responsible for sulfatide binding. Molecular docking suggested that a hydrophilic region, primarily mediated by R42, is responsible for interaction with the sulfatide headgroup, whereas the C-terminal polybasic region contributes to interactions with acyl chains. Furthermore, we demonstrated that, in Dab2 SBP, R42 significantly contributes to the inhibition of platelet P-selectin surface expression. The Dab2 SBP residues that interact with sulfatides resemble those described for sphingolipid-binding in other proteins, suggesting that sulfatide-binding proteins share common binding mechanisms.

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