A first-in-class inhibitor of Hsp110 molecular chaperones of pathogenic fungi.

Proteins of the Hsp110 family are molecular chaperones that play important roles in protein homeostasis in eukaryotes. The pathogenic fungus Candida albicans, which causes infections in humans, has a single Hsp110, termed Msi3. Here, we provide proof-of-principle evidence supporting fungal Hsp110s as targets for the development of new antifungal drugs. We identify a pyrazolo[3,4-b] pyridine derivative, termed HLQ2H (or 2H), that inhibits the biochemical and chaperone activities of Msi3, as well as the growth and viability of C. albicans. Moreover, the fungicidal activity of 2H correlates with its inhibition of in vivo protein folding. We propose 2H and related compounds as promising leads for development of new antifungals and as pharmacological tools for the study of the molecular mechanisms and functions of Hsp110s.

Merging cultures and disciplines to create a drug discovery ecosystem at Virginia commonwealth university: Medicinal chemistry, structural biology, molecular and behavioral pharmacology and computational chemistry.

The Department of Medicinal Chemistry, together with the Institute for Structural Biology, Drug Discovery and Development, at Virginia Commonwealth University (VCU) has evolved, organically with quite a bit of bootstrapping, into a unique drug discovery ecosystem in response to the environment and culture of the university and the wider research enterprise. Each faculty member that joined the department and/or institute added a layer of expertise, technology and most importantly, innovation, that fertilized numerous collaborations within the University and with outside partners. Despite moderate institutional support with respect to a typical drug discovery enterprise, the VCU drug discovery ecosystem has built and maintained an impressive array of facilities and instrumentation for drug synthesis, drug characterization, biomolecular structural analysis and biophysical analysis, and pharmacological studies. Altogether, this ecosystem has had major impacts on numerous therapeutic areas, such as neurology, psychiatry, drugs of abuse, cancer, sickle cell disease, coagulopathy, inflammation, aging disorders and others. Novel tools and strategies for drug discovery, design and development have been developed at VCU in the last five decades; e.g., fundamental rational structure-activity relationship (SAR)-based drug design, structure-based drug design, orthosteric and allosteric drug design, design of multi-functional agents towards polypharmacy outcomes, principles on designing glycosaminoglycans as drugs, and computational tools and algorithms for quantitative SAR (QSAR) and understanding the roles of water and the hydrophobic effect.

Phage-Related Ribosomal Proteases (Prps): Discovery, Bioinformatics, and Structural Analysis.

Many new antimicrobials are analogs of existing drugs, sharing the same targets and mechanisms of action. New antibiotic targets are critically needed to combat the growing threat of antimicrobial-resistant bacteria. Phage-related ribosomal proteases (Prps) are a recently structurally characterized antibiotic target found in pathogens such as Staphylococcus aureus, Clostridioides difficile, and Streptococcus pneumoniae. These bacteria encode an N-terminal extension on their ribosomal protein L27 that is not present in other bacteria. The cleavage of this N-terminal extension from L27 by Prp is necessary to create a functional ribosome. Thus, Prp inhibition may serve as an alternative to direct binding and inhibition of the ribosome. This bioinformatic and structural analysis covers the discovery, function, and structural characteristics of known Prps. This information will be helpful in future endeavors to design selective therapeutics targeting the Prps of important pathogens.

Phage-Related Ribosomal Protease (Prp) of Staphylococcus aureus: In Vitro Michaelis-Menten Kinetics, Screening for Inhibitors, and Crystal Structure of a Covalent Inhibition Product Complex.

Phage-related ribosomal proteases (Prps) are essential for the assembly and maturation of the ribosome in Firmicutes, including the human pathogens Staphylococcus aureus, Streptococcus pneumoniae, and Clostridium difficile. These bacterial proteases cleave off an N-terminal extension of a precursor of ribosomal protein L27, a processing step that is essential for the formation of functional ribosomes. This essential role of Prp in these pathogens has identified this protease as a potential antibiotic target. In this work, we determine the X-ray crystal structure of a covalent inhibition complex at 2.35 Å resolution, giving the first complete picture of the active site of a functional Prp. We also characterize the kinetic activity and screen for potential inhibitors of Prp. This work gives the most complete characterization of the structure and specificity of this novel class of proteases to date.

The Case against Antibiotics and for Anti-Virulence Therapeutics.

Although antibiotics have been indispensable in the advancement of modern medicine, there are downsides to their use. Growing resistance to broad-spectrum antibiotics is leading to an epidemic of infections untreatable by first-line therapies. Resistance is exacerbated by antibiotics used as growth factors in livestock, over-prescribing by doctors, and poor treatment adherence by patients. This generates populations of resistant bacteria that can then spread resistance genes horizontally to other bacterial species, including commensals. Furthermore, even when antibiotics are used appropriately, they harm commensal bacteria leading to increased secondary infection risk. Effective antibiotic treatment can induce bacterial survival tactics, such as toxin release and increasing resistance gene transfer. These problems highlight the need for new approaches to treating bacterial infection. Current solutions include combination therapies, narrow-spectrum therapeutics, and antibiotic stewardship programs. These mediate the issues but do not address their root cause. One emerging solution to these problems is anti-virulence treatment: preventing bacterial pathogenesis instead of using bactericidal agents. In this review, we discuss select examples of potential anti-virulence targets and strategies that could be developed into bacterial infection treatments: the bacterial type III secretion system, quorum sensing, and liposomes.

Molecular Targets and Strategies for Inhibition of the Bacterial Type III Secretion System (T3SS); Inhibitors Directly Binding to T3SS Components.

The type III secretion system (T3SS) is a virulence apparatus used by many Gram-negative pathogenic bacteria to cause infections. Pathogens utilizing a T3SS are responsible for millions of infections yearly. Since many T3SS knockout strains are incapable of causing systemic infection, the T3SS has emerged as an attractive anti-virulence target for therapeutic design. The T3SS is a multiprotein molecular syringe that enables pathogens to inject effector proteins into host cells. These effectors modify host cell mechanisms in a variety of ways beneficial to the pathogen. Due to the T3SS's complex nature, there are numerous ways in which it can be targeted. This review will be focused on the direct targeting of components of the T3SS, including the needle, translocon, basal body, sorting platform, and effector proteins. Inhibitors will be considered a direct inhibitor if they have a binding partner that is a T3SS component, regardless of the inhibitory effect being structural or functional.

Fluorescence Detection of Type III Secretion Using a Glu-CyFur Reporter System in Citrobacter rodentium.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea worldwide. EPEC and the closely related murine model of EPEC infection, Citrobacter rodentium, utilize a type III secretion system (T3SS) to propagate the infection. Since the T3SS is not essential for the bacteria to survive or propagate, inhibiting the virulence factor with a therapeutic would treat the infection without causing harm to commensal bacteria. Studying inhibitors of the T3SS usually requires a BSL-2 laboratory designation and eukaryotic host cells while not indicating the mechanism of inhibition. We have designed a BSL-1 assay using the murine model C. rodentium that does not require mammalian cell culture. This CPG2-reporter assay allows for more rapid analysis of secretion efficiency than Western blotting and is sensitive enough to differentiate between partial and total inhibition of the T3SS. Here we present our method and the results of a small collection of compounds we have screened, including known T3SS inhibitors EGCG, regacin, and aurodox and related quorum sensing inhibitors tannic acid and ellagic acid. We have further characterized EGCG as a T3SS inhibitor and established its IC50 of 1.8 ± 0.4 µM. We also establish tannic acid as a potent inhibitor of the T3SS with an IC50 of 0.65 ± 0.09 µM.

Antibodies Inhibiting the Type III Secretion System of Gram-Negative Pathogenic Bacteria.

Pathogenic bacteria are a global health threat, with over 2 million infections caused by Gram-negative bacteria every year in the United States. This problem is exacerbated by the increase in resistance to common antibiotics that are routinely used to treat these infections, creating an urgent need for innovative ways to treat and prevent virulence caused by these pathogens. Many Gram-negative pathogenic bacteria use a type III secretion system (T3SS) to inject toxins and other effector proteins directly into host cells. The T3SS has become a popular anti-virulence target because it is required for pathogenesis and knockouts have attenuated virulence. It is also not required for survival, which should result in less selective pressure for resistance formation against T3SS inhibitors. In this review, we will highlight selected examples of direct antibody immunizations and the use of antibodies in immunotherapy treatments that target the bacterial T3SS. These examples include antibodies targeting the T3SS of Pseudomonas aeruginosa, Yersinia pestis, Escherichia coli, Salmonella enterica, Shigella spp., and Chlamydia trachomatis.

Delivery of Heterologous Proteins, Enzymes, and Antigens via the Bacterial Type III Secretion System.

The Type III Secretion System (T3SS) is a multimeric protein complex composed of over 20 different proteins, utilized by Gram-negative bacteria to infect eukaryotic host cells. The T3SS has been implicated as a virulence factor by which pathogens cause infection and has recently been characterized as a communication tool between bacteria and plant cells in the rhizosphere. The T3SS has been repurposed to be used as a tool for the delivery of non-native or heterologous proteins to eukaryotic cells or the extracellular space for a variety of purposes, including drug discovery and drug delivery. This review covers the methodology of heterologous protein secretion as well as multiple cases of utilizing the T3SS to deliver heterologous proteins or artificial materials. The research covered in this review will serve to outline the scope and limitations of utilizing the T3SS as a tool for protein delivery.

Animal Models of Type III Secretion System-Mediated Pathogenesis.

The type III secretion system (T3SS) is a conserved virulence factor used by many Gram-negative pathogenic bacteria and has become an important target for anti-virulence drugs. Most T3SS inhibitors to date have been discovered using in vitro screening assays. Pharmacokinetics and other important characteristics of pharmaceuticals cannot be determined with in vitro assays alone. In vivo assays are required to study pathogens in their natural environment and are an important step in the development of new drugs and vaccines. Animal models are also required to understand whether T3SS inhibition will enable the host to clear the infection. This review covers selected animal models (mouse, rat, guinea pig, rabbit, cat, dog, pig, cattle, primates, chicken, zebrafish, nematode, wax moth, flea, fly, and amoeba), where T3SS activity and infectivity have been studied in relation to specific pathogens (Escherichia coli, Salmonella spp., Pseudomonas spp., Shigella spp., Bordetella spp., Vibrio spp., Chlamydia spp., and Yersinia spp.). These assays may be appropriate for those researching T3SS inhibition.

Natural Product Type III Secretion System Inhibitors.

Many known inhibitors of the bacterial type III secretion system (T3SS), a virulence factor used by pathogenic bacteria to infect host cells, are natural products. These compounds, produced by bacteria, fungi, and plants, may have developed as prophylactic treatments for potential attack by bacterial pathogens or as an attempt by symbiotic organisms to protect their hosts. Regardless, better understanding of the structures and mechanisms of action of these compounds may open opportunities for drug development against diseases caused by pathogens utilizing the T3SS. This review will cover selected known natural products of the T3SS and detail what is known of their origin and mechanism of action. These inhibitors highlight nature's ability to modulate interactions between organisms at a cellular level.

Molecular insights into the biosynthesis of guadinomine: a type III secretion system inhibitor.

Guadinomines are a recently discovered family of anti-infective compounds produced by Streptomyces sp. K01-0509 with a novel mode of action. With an IC(50) of 14 nM, guadinomine B is the most potent known inhibitor of the type III secretion system (TTSS) of Gram-negative bacteria. TTSS activity is required for the virulence of many pathogenic Gram-negative bacteria including Escherichia coli , Salmonella spp., Yersinia spp., Chlamydia spp., Vibrio spp., and Pseudomonas spp. The guadinomine (gdn) biosynthetic gene cluster has been cloned and sequenced and includes 26 open reading frames spanning 51.2 kb. It encodes a chimeric multimodular polyketide synthase, a nonribosomal peptide synthetase, along with enzymes responsible for the biosynthesis of the unusual aminomalonyl-acyl carrier protein extender unit and the signature carbamoylated cyclic guanidine. Its identity was established by targeted disruption of the gene cluster as well as by heterologous expression and analysis of key enzymes in the biosynthetic pathway. Identifying the guadinomine gene cluster provides critical insight into the biosynthesis of these scarce but potentially important natural products.

Total synthesis of (-)-callipeltoside A.

A total synthesis of (-)-callipeltoside A (1) has been achieved. The core macrocycle was made via a dual macrolactonization/pyran hemiketal formation reaction, developed to circumvent issues related to the reversible nature of acylketene formation from ß-keto lactone substrates. Initial approaches to the core of the natural product that revolved around ring-closing metathesis (RCM) and relay ring-closing metathesis (RRCM) reactions are also described.

Room temperature acylketene formation? 1,3-Dioxin-4-ones via silver(I) activation of phenylthioacetoacetate in the presence of ketones.

Silver(I) activation of thioacetoacetates in the presence of ketones produces 1,3-dioxin-4-ones. Mechanistic studies addressing the intermediacy of an acylketene intermediate are described.

A Useful Modification of the Evans Magnesium Halide-Catalyzed anti-Aldol Reaction: Application to Enolizable Aldehydes.

A practical protocol for use of the magnesium halide-catalyzed anti-aldol reaction of an Evans N-acyloxazolidinone with enolizable aldehydes is reported. The yields of anti-aldol adducts for saturated or unsaturated and branched or unbranched aliphatic aldehydes are preparatively useful.

Decarboxylative isomerization of N-Acyl-2-oxazolidinones to 2-oxazolines.

N-Acyl-2-oxazolidinones are ring-opened by lithium iodide and decarboxylated in the presence of a mild proton source. Further reaction with an amine base provides 2-oxazolines. The transformation is general for oxazolidinones unsubstituted in the 5 position and occurs under mild conditions (25-50 degrees C). These results complement the existing methods for this transformation by allowing lower temperatures and/or avoiding metal catalysts.