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1. Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence Compounds

   https://doi.org/10.1128/spectrum.03877-22  

   Dye, Keane J. ; Vogelaar, Nancy J. ; O’Hara, Megan ; Sobrado, Pablo ; Santos, Webster ; Carlier, Paul R. ; Yang, Zhaomin ; Lostroh, Phoebe ( December 2022 , Microbiology Spectrum)

   Visca, Paolo (Ed.)

   ABSTRACT 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. 

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2. Identifying Small Molecules That Promote Quasipalindrome-Associated Template-Switch Mutations in Escherichia coli

   https://doi.org/10.1534/g3.120.401106  

   Klaric, Julie A. ; Perr, Eli L. ; Lovett, Susan T. ( May 2020 , G3: Genes|Genomes|Genetics)

   DNA can assemble into non-B form structures that stall replication and cause genomic instability. One such secondary structure results from an inverted DNA repeat that can assemble into hairpin and cruciform structures during DNA replication. Quasipalindromes (QP), imperfect inverted repeats, are sites of mutational hotspots. Quasipalindrome-associated mutations (QPMs) occur through a template-switch mechanism in which the replicative polymerase stalls at a QP site and uses the nascent strand as a template instead of the correct template strand. This mutational event causes the QP to become a perfect or more perfect inverted repeat. Since it is not fully understood how template-switch events are stimulated or repressed, we designed a high-throughput screen to discover drugs that affect these events. QP reporters were engineered in the Escherichia coli lacZ gene to allow us to study template-switch events specifically. We tested 700 compounds from the NIH Clinical Collection through a disk diffusion assay and identified 11 positive hits. One of the hits was azidothymidine (zidovudine, AZT), a thymidine analog and DNA chain terminator. The other ten were found to be fluoroquinolone antibiotics, which induce DNA-protein crosslinks. This work shows that our screen is useful in identifying small molecules that affect quasipalindrome-associated template-switch mutations. We are currently assessing more small molecule libraries and applying this method to study other types of mutations. 

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3. Combinatorial phenotypic screen uncovers unrecognized family of extended thiourea inhibitors with copper-dependent anti-staphylococcal activity

   https://doi.org/10.1039/c6mt00003g  

   Dalecki, Alex G. ; Malalasekera, Aruni P. ; Schaaf, Kaitlyn ; Kutsch, Olaf ; Bossmann, Stefan H. ; Wolschendorf, Frank ( February 2016 , Metallomics)

   The continuous rise of multi-drug resistant pathogenic bacteria has become a significant challenge for the health care system. In particular, novel drugs to treat infections of methicillin-resistant Staphylococcus aureus strains (MRSA) are needed, but traditional drug discovery campaigns have largely failed to deliver clinically suitable antibiotics. More than simply new drugs, new drug discovery approaches are needed to combat bacterial resistance. The recently described phenomenon of copper-dependent inhibitors has galvanized research exploring the use of metal-coordinating molecules to harness copper’s natural antibacterial properties for therapeutic purposes. Here, we describe the results of the first concerted screening effort to identify copper-dependent inhibitors of Staphylococcus aureus. A standard library of 10 000 compounds was assayed for anti-staphylococcal activity, with hits defined as those compounds with a strict copper-dependent inhibitory activity. A total of 53 copper-dependent hit molecules were uncovered, similar to the copper independent hit rate of a traditionally executed campaign conducted in parallel on the same library. Most prominent was a hit family with an extended thiourea core structure, termed the NNSN motif. This motif resulted in copper-dependent and copper-specific S. aureus inhibition, while simultaneously being well tolerated by eukaryotic cells. Importantly, we could demonstrate that copper binding by the NNSN motif is highly unusual and likely responsible for the promising biological qualities of these compounds. A subsequent chemoinformatic meta-analysis of the ChEMBL chemical database confirmed the NNSNs as an unrecognized staphylococcal inhibitor, despite the family’s presence in many chemical screening libraries. Thus, our copper-biased screen has proven able to discover inhibitors within previously screened libraries, offering a mechanism to reinvigorate exhausted molecular collections.

    

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4. Phenotypic Screening of Prospective Analgesics Among FDA‐Approved Compounds using an iPSC‐Based Model of Acute and Chronic Inflammatory Nociception

   https://doi.org/10.1002/advs.202303724  

   Black, Bryan James ; Ghazal, Rasha El ; Lojek, Neal ; Williams, Victoria ; Rajput, Jai Singh ; Lawson, Jennifer M. ( January 2024 , Advanced Science)

   Classical target‐based drug screening is low‐throughput, largely subjective, and costly. Phenotypic screening based on in vitro models is increasingly being used to identify candidate compounds that modulate complex cell/tissue functions. Chronic inflammatory nociception, and subsequent chronic pain conditions, affect peripheral sensory neuron activity (e.g., firing of action potentials) through myriad pathways, and remain unaddressed in regard to effective, non‐addictive management/treatment options. Here, a chronic inflammatory nociception model is demonstrated based on induced pluripotent stem cell (iPSC) sensory neurons and glia, co‐cultured on microelectrode arrays (MEAs). iPSC sensory co‐cultures exhibit coordinated spontaneous extracellular action potential (EAP) firing, reaching a stable baseline after ≈27 days in vitro (DIV). Spontaneous and evoked EAP metrics are significantly modulated by 24‐h incubation with tumor necrosis factor‐alpha (TNF‐α), representing an inflammatory phenotype. Compared with positive controls (lidocaine), this model is identified as an “excellent” stand‐alone assay based on a modified Z’ assay quality metric. This model is then used to screen 15 cherry‐picked, off‐label, Food and Drug Administration (FDA)‐approved compounds; 10 of 15 are identified as “hits”. Both hits and “misses” are discussed in turn. In total, this data suggests that iPSC sensory co‐cultures on MEAs may represent a moderate‐to‐high‐throughput assay for drug discovery targeting inflammatory nociception.

    

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5. Identifying Inhibitors Targeting the Nonstructural Protein 15 and Main Protease of Coronaviruses Using Molecular Docking and Molecular Dynamics Simulation

   Webber, Nakoa K. ( January 2021 , Rowan digital works)

   The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2020 has impacted daily life globally for over a year. While multiple vaccines have been authorized for emergency use and one oral medication has entered clinical trials, we are still seeking antiviral drugs for a long-term treatment for SARS-CoV-2 as well as other coronaviruses. Computational drug screenings of two SARS-CoV-2 protein target candidates are presented in this thesis: the nidoviral RNA uridylate-specific endoribonuclease (Nsp15) and the main protease (Mpro) of SARS-CoV-2. Nonstructural proteins of coronaviruses were selected as targets as they are more conserved across coronavirus strains than structural proteins. High throughput virtual screening of small molecule libraries including DrugBank and ZINC 15 resulted in several promising compounds for each of these targets. Molecular dynamics simulation allowed us to predict the binding energies for these compounds using molecular mechanics with generalized born surface area solvation calculations (MM-GBSA). Four top compounds were discovered for Nsp15 and eight compounds for Mpro. 

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