Solubility is a key metric for therapeutic compounds. Conversely, insoluble compounds cloud the accuracy of assays at all stages of chemical biology and drug discovery. Herein, we disclose naïve Bayesian classifier models to predict aqueous solubility. Publicly accessible aqueous solubility data were used to create two full, or nonpruned, training sets. These two sets were also combined to create a full fused set, and a training set comprised of a literature collation of solubility data was also considered as a reference. We tested different extents of data pruning on the training sets and constructed machine learning models that were evaluated with two independent, external test sets that contained compounds that were different from the training sets. The best pruned and fused model was significantly more accurate, in comparison to either the full model or the full fused model, with the prediction of these external test sets. By carefully removing data from the training set, less information can be used to create more accurate machine learning models for aqueous solubility. This knowledge and the curated training sets should prove useful to future machine learning approaches.

Published Mycobacterium tuberculosis β-ketoacyl-ACP synthase KasA inhibitors lack sufficient potency and/or pharmacokinetic properties. A structure-based approach was used to optimize existing KasA inhibitor DG167. This afforded indazole JSF-3285 with a 30-fold increase in mouse plasma exposure. Biochemical, genetic, and X-ray studies confirmed JSF-3285 targets KasA. JSF-3285 offers substantial activity in an acute mouse model of infection and in the corresponding chronic infection model, with efficacious reductions in colony-forming units at doses as low as 5 mg/kg once daily orally and improvement of the efficacy of front-line drugs isoniazid or rifampicin. JSF-3285 is a promising preclinical candidate for tuberculosis.

The diffusible signal factors (DSFs) are a family of quorum-sensing autoinducers (AIs) produced and detected by numerous gram-negative bacteria. The DSF family AIs are fatty acids, differing in their acyl chain length, branching, and substitution but having in common a cis-2 double bond that is required for their activity. In both human and plant pathogens, DSFs regulate diverse phenotypes, including virulence factor expression, antibiotic resistance, and biofilm dispersal. Despite their widespread relevance to both human health and agriculture, the molecular basis of DSF recognition by their cellular receptors remained a mystery. Here, we report the first structure-function studies of the DSF receptor regulation of pathogenicity factor R (RpfR). We present the X-ray crystal structure of the RpfR DSF-binding domain in complex with the Burkholderia DSF (BDSF), which to our knowledge is the first structure of a DSF receptor in complex with its AI. To begin to understand the mechanistic role of the BDSF-RpfR contacts observed in the biologically important complex, we have also determined the X-ray crystal structure of the RpfR DSF-binding domain in complex with the inactive, saturated isomer of BDSF, dodecanoic acid (C12:0). In addition to these ligand-receptor complex structures, we report the discovery of a previously overlooked RpfR domain and show that it binds to and negatively regulates the DSF synthase regulation of pathogenicity factor F (RpfF). We have named this RpfR region the RpfF interaction (FI) domain, and we have determined its X-ray crystal structure alone and in complex with RpfF. These X-ray crystal structures, together with extensive complementary in vivo and in vitro functional studies, reveal the molecular basis of DSF recognition and the importance of the cis-2 double bond to DSF function. Finally, we show that throughout cellular growth, the production of BDSF by RpfF is post-translationally controlled by the RpfR N-terminal FI domain, affecting the cellular concentration of the bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Thus, in addition to describing the molecular basis for the binding and specificity of a DSF for its receptor, we describe a receptor-synthase interaction regulating bacterial quorum-sensing signaling and second messenger signal transduction.

The triazine antitubercular JSF-2019 was of interest due to its in vitro efficacy and the nitro group shared with the clinically relevant delamanid and pretomanid. JSF-2019 undergoes activation requiring F420H2 and one or more nitroreductases in addition to Ddn. An intrabacterial drug metabolism (IBDM) platform was leveraged to demonstrate the system kinetics, evidencing formation of NO⋅ and a des-nitro metabolite. Structure-activity relationship studies focused on improving the solubility and mouse pharmacokinetic profile of JSF-2019 and culminated in JSF-2513, relying on the key introduction of a morpholine. Mechanistic studies with JSF-2019, JSF-2513, and other triazines stressed the significance of achieving potent in vitro efficacy via release of intrabacterial NO⋅ along with inhibition of InhA and, more generally, the FAS-II pathway. This study highlights the importance of probing IBDM and its potential to clarify mechanism of action, which in this case is a combination of NO⋅ release and InhA inhibition.

Cell wall biosynthesis inhibitors have proven highly effective for treating tuberculosis (TB). We discovered and validated members of the indazole sulfonamide class of small molecules as inhibitors of Mycobacterium tuberculosis KasA—a key component for biosynthesis of the mycolic acid layer of the bacterium’s cell wall and the same pathway as that inhibited by the first-line antitubercular drug isoniazid (INH). One lead compound, DG167, demonstrated synergistic lethality in combination with INH and a transcriptional pattern consistent with bactericidality and loss of persisters. Our results also detail a novel dual-binding mechanism for this compound as well as substantial structure-activity relationships (SAR) that may help in lead optimization activities. Together, these results suggest that KasA inhibition, specifically, that shown by the DG167 series, may be developed into a potent therapy that can synergize with existing antituberculars.

Mycobacterium tuberculosis infection is responsible for a global pandemic. New drugs are needed that do not show cross-resistance with the existing front-line therapeutics. A triazine antitubercular hit led to the design of a related pyrimidine family. The synthesis of a focused series of these analogs facilitated exploration of their in vitro activity, in vitro cytotoxicity, and physiochemical and absorption-distribution-metabolism-excretion properties. Select pyrimidines were then evaluated for their pharmacokinetic profiles in mice. The findings suggest a rationale for the further evolution of this promising series of antitubercular small molecules, which appear to share some similarities with the clinical compound PA-824 in terms of activation, while highlighting more general guidelines for the optimization of small-molecule antitubercular agents.

The flippase MurJ is responsible for transporting the cell wall intermediate lipid II from the cytoplasm to the outside of the cell. While essential for the survival of bacteria, it remains an underexploited target for antibacterial therapy. The humimycin antibiotics are lipid II flippase (MurJ) inhibitors that were synthesized on the basis of bioinformatic predictions derived from secondary metabolite gene clusters found in the human microbiome. Here, we describe an SAR campaign around humimycin A that produced humimycin 17S. Compared to humimycin A, 17S is a more potent β-lactam potentiator, has a broader spectrum of activity, which now includes both methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE), and did not lead to any detectable resistance when used in combination with a β-lactam. Combinations of β-lactam and humimycin 17S provide a potentially useful long-term MRSA regimen.

Bacterial culture broth extracts have been the starting point for the development of numerous therapeutics. However, only a small fraction of bacterial biosynthetic diversity is accessible using this strategy. Here, we apply a discovery approach that bypasses the culturing step entirely by bioinformatically predicting small molecule structures from the primary sequences of the biosynthetic gene clusters. These structures are then chemically synthesized to give synthetic-bioinformatic natural products (syn-BNPs). Using this approach, we screened syn-BNPs inspired by nonribosomal peptide synthetases against microbial pathogens, and discovered an antibiotic for which no resistance could be identified and an antifungal agent with activity against diverse fungal pathogens.

The synthetic bioinformatic natural products (syn-BNPs) approach identifies putative natural products that are validated directly by independent synthesis. Its application led to the identification of humimycins, non-ribosomal peptides that have antimicrobial activity in mice. Here we present a natural product discovery approach, whereby structures are bioinformatically predicted from primary sequence and produced by chemical synthesis (synthetic-bioinformatic natural products, syn-BNPs), circumventing the need for bacterial culture and gene expression. When we applied the approach to nonribosomal peptide synthetase gene clusters from human-associated bacteria, we identified the humimycins. These antibiotics inhibit lipid II flippase and potentiate β-lactam activity against methicillin-resistant Staphylococcus aureus in mice, potentially providing a new treatment regimen.

OF THE DISSERTATION.........................................................................................ii ACKNOWLEDGEMENTS.............................................................................................................iv DEDICATION..................................................................................................................................v TABLE OF CONTENTS.................................................................................................................vi LIST OF TABLES........................................................................................................................viii LIST OF FIGURES.........................................................................................................................ix LIST OF SCHEMES.....................................................................................................................xii ABBREVIATIONS.......................................................................................................................xiv CHAPTER ONE...............................................................................................................................

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1-Nrf2 protein-protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure-activity relationships support its use as a lead for our ongoing optimization.

Activation of the antioxidant response element (ARE) upregulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein Keap1 and the transcription factor Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small-molecule inhibitors of the Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance assay. The FP assay exhibits considerable tolerance toward DMSO and produced a Z′ factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high-throughput screening assay to discover small-molecule inhibitors of Keap1-Nrf2 interaction.

Prodrug design is an important part of drug discovery. Prodrugs can offer many advantages over parent drugs such as increased solubility, enhanced stability, improved bioavailability, reduced side effects, and better selectivity. Many prodrugs have been used successfully in the clinic; examples include oseltamivir in anti-influenza therapy, enalapril in anti-hypertension therapy, capecitabine in cancer therapy, and omeprazole in the treatment of peptic ulcer. A key step in prodrug design is the incorporation of an activation mechanism that can convert the prodrug into the active species in an efficient and/or controlled manner to meet the needs of a given medical application. Prodrug activation can be achieved through enzyme-mediated hydrolytic or oxidoreductive processes while activation of some prodrugs may proceed through pure chemical nonenzymatic processes. This review focuses on the hydrolytic enzymes that have been used in prodrug activation, including transferases, hydrolases, and lyases.