Four known small molecule uracil-DNA glycosylase (UNG) inhibitors were synthesized and tested against human melanoma cells, IgR3 and MM200. They were found to be effective against cell proliferation at micromolar concentrations and to operate through a nonapoptotic mechanism. Thus, small molecules that target UNG may be useful as potential chemotherapeutic agents against human melanoma.

Indiana University-Purdue University Indianapolis (IUPUI) / The human aldehyde dehydrogenase superfamily consists of 19 distinct genetic loci that play key roles in both health and disease. Aldehyde dehydrogenases are primarily involved in the metabolism of reactive aldehyde substrates; the ALDH1A subfamily, in particular, metabolizes retinaldehyde and is involved in a pathway regulating tissue differentiation, cell proliferation, and apoptosis. Recently, ALDH1 isoenzymes have been implicated as significant elements in cancer progression. ALDH1 activity has been used as a marker of cancer stem cells, a subpopulation of cancer stem cells with high drug resistance, proliferative potential, and ability to differentiate into multiple cell types. In accordance with this, ALDH1 activity and expression has been shown to correlate with lower survival, increased chemoresistance, and increased chance of relapse in multiple solid cancer types, including breast, ovarian, lung, and colorectal. Despite the clear relevance of ALDH1 enzymes in cancer, the specific roles of individual isoenzymes are unclear. Isoenzyme-selective small molecule modulators of the ALDH1A subfamily would allow the probing of the function of individual isoenzymes in healthy and disease states. Two ALDH1A1 inhibitors, CM38 and C10, were previously identified in a high-throughput screen. In this study, CM38, an ALDH1A1-selective inhibitor, and CM10, an ALDH1A inhibitor, were characterized using kinetic assays, structural biology, and cell culture experiments. A structure-activity relationship was built for each series, and an X-ray crystallography structure was used to determine the binding mode. These approaches allowed the investigation of the ALDH1A active site and identification of structural features that can be used to design and improve selective modulators of this subfamily. CM38 and CM10 were also tested in a breast cancer cell line to determine their efficacy in a cellular environment. While the CM38 series showed warning signs of potential off-target toxicity, members of the CM10 compound series showed excellent initial characteristics as potential chemical tools. The results of this study may be useful in the design of new chemical tools to delineate the functions of individual ALDH1 isoenzymes in cancer biology, as well as in the development of drugs to selectively target cancer stem cells.

Chlamydia are obligate intracellular bacterial pathogens that are responsible for infectious blindness, sexually transmitted infections, and acute respiratory disease in humans. These pathogens undergo an essential biphasic developmental cycle differentiating between two functionally distinct forms known as the infectious elementary body (EB) and the replicative reticulate body (RB). Identifying the signals and regulatory mechanisms that enable Chlamydia to establish infection, differentiate between the two developmental forms, and survive within the host cell is critical to understanding chlamydial pathogenesis and developing future therapeutic strategies. In pathogenic bacteria, serine, threonine, and tyrosine (Ser/Thr/Tyr) protein kinases and phosphatases are critical for development, metabolism, and virulence. Chlamydia encode two validated protein kinases (pkn1 and pknd), a putative protein phosphatase (ctl0511; CppA), and appear capable of global phosphorylation that differs between the developmental forms. While these findings support a role for protein phosphorylation in chlamydial pathogenesis, a validated cognate protein phosphatase for Pkn1 and PknD mediating reversible phosphorylation was lacking. We hypothesized that CppA is the partner phosphatase for the chlamydial protein kinases, and in this study we validated and characterized CppA as a broad specificity protein phosphatase type 2C. Using in vivo and in vitro approaches we demonstrated that CppA acts on P-Ser/Thr/Tyr residues and can dephosphorylate multiple chlamydial protein substrates including PknD and the FHA 2 domain of CdsD, a component of the type 3 secretion apparatus. The importance of CppA for chlamydial growth and development was determined using a chemical “knock-out” approach and study of CppA missense mutations identified in slow growing C. trachomatis L2 chemical mutants. Treatment of C. trachomatis L2, C. trachomatis D, and C. muridarum with CppA inhibitors significantly reduced progeny levels and inclusion size in a time dependent manner with more significant growth inhibition in the first 12 hours post infection. Collectively, our findings support that CppA works in conjunction with PknD, and likely Pkn1, to mediate reversible phosphorylation of multiple protein substrates leading to changes in chlamydial physiology that appear to be key for early steps in development.

Discovered in 1980, HTLV-1 (Human T-cell Leukemia Virus-1), was the first identified human retrovirus and is shown to be associated with a variety of diseases including: adult T-cell leukemia lymphoma (ATLL), tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM), chronic arthropathy, uveitis, infective dermatitis, and polymyositis. The mechanism by which the virus causes disease is still unknown. HTLV- 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan, the Caribbean basin, Central and West Africa, the Southeastern United States, Melanesia, parts of South Africa, the Middle East and India. Approximately 30 million people are infected by HTLV-1 worldwide, although only 3-5% of the infected individuals evolve Adult T-cell Leukemia (ATL) during their life and the prognosis for those infected is still poor. The retroviral proteases (PRs) are essential for viral replication because they process viral Gag and Gag-(Pro)-Pol polyproteins during maturation, much like the PR from Human Immunodeficiency Virus-1 (HIV-1). Various antiviral inhibitors are in clinical use and one of the most significant classes is HIV-1 PR inhibitors, which have used for antiretroviral therapy in the treatment of AIDS. HTLV-1 PR and HIV-1 PR are homodimeric aspartic proteases with 125 and 99 residues, respectively. Even though substrate specificities of these two enzymes are different, HTLV-1 PR shares 28% similarity with HIV-1 PR overall and the substrate binding sites have 45% similarity. In addition to the 125-residue full length HTLV-1 PR, constructs with various C- terminal deletions (giving proteases with lengths of 116, 121, or 122 amino acids) were made in order to elucidate the effect of the residues in the C-terminal region. It was suggested that five amino acids in the C-terminal region are not necessary for the enzymatic activity in Hayakawa et al. 1992. In 2004 Herger et al. had suggested that 10 amino acids at the C-terminal region are not necessary for catalytic activity. A recent paper suggested that C-terminal residues are essential; and that catalytic activity lowers upon truncation, with even the last 5 amino acids necessary for full catalytic activity (1). The mutation L40I has been made to prevent autoproteolysis and the W98V mutation was made to make the active site of HTLV-1 PR similar to HIV-1 PR. We have characterized C-terminal amino acids of HTLV-1 PR as not being essential for full catalytic activity. We have discovered potential new inhibitors by in silico screening of 116-HTLV-1 PR. These small molecules were tested kinetically for various constructs including the 116, 121 and 122-amino acid forms of HTLV-1 PR. Inhibitors with the best inhibition constants were used in HTLV-1 infected cells and one of the inhibitors seems to inhibit gag processing.

The human body is colonized by more than 100 trillion microbes which make up an essential part of the body and plays a significant role in health. We now know the over use and misuse of broad-spectrum antibiotics can disrupt this microbiome contributing to the onset of disease and runs the risk of promoting antibiotic resistance. With antibiotic research still on the decline, new strategies are greatly needed to combat emerging pathogens while maintaining a healthy microbiome. We therefore set out to present a novel species-selective antimicrobial drug discovery strategy. Disruption of the homeostasis within the oral cavity can trigger the onset of one of the most common bacterial infections, periodontal disease. Even though the oral cavity is one of the most diverse sites on the human body, the Gram-negative colonizer, Porphyromonas gingivalis has long been considered a key player in the initiation of periodontitis, suggesting the potential for novel narrow-spectrum therapeutics. By targeting key pathogens, it may be possible to treat periodontitis while allowing for the recolonization of the beneficial, healthy flora. Therefore, we set out to use P. gingivalis and periodontal disease as a model for pathogen-specific antimicrobial drug discovery. In this study we present a unique approach to predict essential gene targets selective for the periodontal pathogen within the oral environment. Using our knowledge of metabolic networks and essential genes we identified a “druggable” essential target, meso-diaminopimelate dehydrogenase, which is found in a limited number of species. This enzyme, meso-diaminopimelate dehydrogenase from P. gingivalis, was first expressed and purified, then characterized for enzymatic inhibitor screening studies. We then applied a computer-based drug discovery method, combining pharmacophore models, high-throughput virtual screening and molecular docking. Utilizing the ZINC database we virtually screened over 9 million small-molecules to identify several potential target-specific inhibitors. Finally, we used target-based and whole-cell based biochemical screening to assess in vitro activity. We conclude that the establishment of this target and screening strategy provides a framework for the future development of new antimicrobials and drug discovery.

SecA ATPase is a critical member of the Sec system, which is important in the translocation of membrane and secreted polypeptides/proteins in bacteria. Small molecule inhibitors can be very useful research tools as well as leads for future antimicrobial agent development. Based on previous virtual screening work, we optimized the structures of two hit compounds and obtained SecA ATPase inhibitors with IC50 in the single digit micromolar range. These represent the first low micromolar inhibitors of bacterial SecA and will be very useful for mechanistic studies. Post synthetic modification is an important and efficient way of DNA functionalization especially in DNA aptamer selection. In this research, the feasibility of norbornene (Neo) modified thymidine triphosphate incorporation was described. Besides, substituted tetrazines have been found to undergo facile inversed electron demand Diels-Alder reactions with "tunable" reaction rates. This finding paves the way to utilize tetrazine conjugation reactions for not only DNA but also other labeling work.

<p>Computational protein design aims at identifying protein mutations and conformations with desired target properties (such as increased protein stability, switch of substrate specificity, or novel function) from a vast combinatorial space of candidate solutions. The development of algorithms to efficiently and accurately solve problems in protein design has thus posed significant computational and modeling challenges. Despite the inherent hardness of protein design, a number of computational techniques have been previously developed and applied to a wide range of protein design problems. In many cases, however, the available computational protein design techniques are deficient both in computational power and modeling accuracy. Typical simplifying modeling assumptions for computational protein design are the rigidity of the protein backbone and the discretization of the protein side-chain conformations. Here, we present the derivation, proofs of correctness and complexity, implementation, and application of novel algorithms for computational protein design that, unlike previous approaches, have provably-accurate guarantees even when backbone or continuous side-chain flexibility are incorporated into the model. We also describe novel divide-and-conquer and dynamic programming algorithms for improved computational efficiency that are shown to result in speed-ups of up to several orders of magnitude as compared to previously-available techniques. Our novel algorithms are further incorporated as part of K*, a provably-accurate ensemble-based algorithm for protein-ligand binding prediction and protein design. The application of our suite of protein design algorithms to a variety of problems, including enzyme redesign and small-molecule inhibitor design, is described. Experimental validation, performed by our collaborators, of a set of our computational predictions confirms the feasibility and usefulness of our novel algorithms for computational protein design.</p> / Dissertation

Abstract Macrophages found in the tumor microenvironment play a crucial role in initiating an immunosuppressive tumor microenvironment that negatively impacts immunotherapy efficacy and aids tumor progression and metastasis. Constituting the most abundant immune cell in tumor microenvironment (TME), tumor associated macrophages (TAMs) have emerged as an attractive approach for anti-cancer therapy. However, two major challenges need to be overcome for successfully utilizing macrophages for immunotherapy. First, tumors repolarize the TAMs predominantly to M2 tumor-aiding phenotype by secreting various immunosuppressive cytokines. Second, cancer cells overexpress a membrane protein CD47 that interacts with signal-regulating protein alpha (SIRPalpha) expressed on macrophages. This crosstalk provides a downregulatory signal in the form of activation of SHP1/2 that inhibits cancer cell phagocytosis, and CD47, therefore, functions as a “don’t-eat-me” signal. We rationalized that these challenges can be overcome by engineering a nanoparticle system that can deliver a rationale combination of immunomodulatory agents to the TAMs that can both repolarize the M2 macrophages to M1 phenotype efficiently and concurrently block CD47-SIRPalpha interactions by inhibiting SHP2 signaling. Herein, we designed a lipid nanoparticle (LNP) system loaded with amphiphilic R848-cholesterol in its hydrophobic lipid bilayer, while SHP099 gets encapsulated in the hydrophilic core. Our previous studies have shown that the conjugation of cholesterol to the inhibitor stabilizes the lipid bilayer at a high inhibitor concentration. The LNPs showed high optimal drug loading, size, and stability. In vitro studies showed that the M2 macrophages treated with the LNPs system repolarized to M1 phenotype and expressed co-stimulatory molecules while having enhanced phagocytic potential. In vivo efficacy studies in 4T1 tumor-bearing mice showed that LNPs exhibit superior anti-tumor efficacy compared to other treatments. We evaluated the effect of MARCO-targeted LPNs by the conjugating anti-MARCO antibody on the LPN surface. However, no comparable difference in treatment efficacy was observed between the targeted MARCO-LNPs and the non-targeted LNPs. These results demonstrate that the MARCO targeting system designed in this study is largely ineffective in the targeted delivery of its drug cargo specifically to TAMs. Thus, the lipid nanoparticle-mediated co-delivery of a rational combination of TLR7/8 agonist and SHP2 inhibitor in the TAMs increases M2 to M1 repolarization and phagocytosis potential of macrophages. Recommended Citation Malik, V., Ramesh, A. and Kulkarni, A.A. (2021), TLR7/8 Agonist and SHP2 Inhibitor Loaded Nanoparticle Enhances Macrophage Immunotherapy Efficacy. Adv. Therap., 4: 2100086. https://doi.org/10.1002/adtp.202100086

The alarming rise of antibacterial drug resistance and the dwindling supply of novel antibiotics highlight the need for innovative approaches in combating bacterial infections. Traditionally, antibacterial drug discovery campaigns have largely been conducted in rich media. Such growth conditions are not representative of the host environment and render many metabolic pathways, otherwise needed for survival and infection, dispensable. Such pathways have been overlooked in conventional drug discovery campaigns despite their validity as potential antibacterial targets. The work presented in this thesis focuses on the development and validation of a screening strategy for the identification and mechanism of action determination of novel inhibitors of metabolic pathways in bacteria under nutrient-limited conditions. This screen led to the identification of MAC168425, MAC173979 and MAC13772 as inhibitors that target glycine metabolism, p-aminobenzoic acid biosynthesis and biotin biosynthesis, respectively. Moreover, it established this approach as a general platform that can be applied for different organisms with synthetic or natural product libraries. Additional mechanistic studies of the biotin biosynthesis inhibitor, MAC13772, resulted in solving the crystal structure of BioA in complex with MAC13772. Analysis of the co-structure confirmed our proposed mode of inhibition and provided information for strategies for rational drug design. Investigation of the antibacterial activity of MAC13772 revealed its potency against a number of pathogens. Furthermore, we show how MAC13772 acts synergistically with rifampicin in clearing growing mycobacterial cultures. The potential of this inhibitor as a lead for preclinical pharmacokinetic studies and for antibacterial drug development is discussed. We also discuss our current efforts to develop a metabolomic platform for the characterization of novel antibacterials that can be used in concert with our current approach to chart the metabolic response of bacteria to chemical perturbants and to generate testable hypotheses regarding the mode of action of novel inhibitors of bacterial metabolism. / Thesis / Doctor of Philosophy (PhD)

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