Empiric antimicrobial therapy often consists of the combination of Gram-positive coverage with vancomycin (VAN) and Gram-negative coverage, specifically an anti-pseudomonal beta-lactam, such as piperacillin-tazobactam (PTZ). Nephrotoxicity is commonly associated with VAN therapy; however, recent reports demonstrate increasing nephrotoxicity rates among patients treated with the combination of VAN and PTZ. This study evaluated the effect of the VAN/PTZ combination on acute kidney injury (AKI), as defined by the RIFLE criteria, compared to VAN and PTZ monotherapies. Overall, 11,650 patients were analyzed, with 1,647 (14.1%) AKI cases occurring. AKI was significantly more frequent in the VAN/PTZ group (21%) compared to either monotherapy group (VAN 8.3%, PTZ 7.8%, p<0.001 for both). Combination therapy was independently associated with higher AKI odds compared to monotherapy with either agent (aOR=2.03; 95% CI 1.74-2.39; aOR=2.31; 95% CI 1.97-2.71, for VAN and PTZ, respectively). Receipt of concomitant nephrotoxic drugs were independently associated with increased AKI rates, as were increased duration of therapy, length of hospital stay, increasing severity of illness, and increasing baseline renal function. VAN combined with PTZ was associated with twice the odds of AKI development compared to either agent as monotherapy. This demonstrates the need for judicious use of combination empiric therapy.

Methyl group transfer from S-adenosyl-l-methionine (AdoMet) to various substrates including DNA, proteins, and natural products (NPs), is accomplished by methyltransferases (MTs). Analogs of AdoMet, bearing an alternative S-alkyl group can be exploited, in the context of an array of wild-type MT-catalyzed reactions, to differentially alkylate DNA, proteins, and NPs. This technology provides a means to elucidate MT targets by the MT-mediated installation of chemoselective handles from AdoMet analogs to biologically relevant molecules and affords researchers a fresh route to diversify NP scaffolds by permitting the differential alkylation of chemical sites vulnerable to NP MTs that are unreactive to traditional, synthetic organic chemistry alkylation protocols. The full potential of this technology is stifled by several impediments largely deriving from the AdoMet-based reagents, including the instability, membrane impermeability, poor synthetic yield and resulting diastereomeric mixtures. To circumvent these main liabilities, novel chemoenzymatic strategies that employ methionine adenosyltransferases (MATs) and methionine (Met) analogs to synthesize AdoMet analogs in vitro were advanced. Unstable AdoMet analogs are simultaneously utilized in a one-pot reaction by MTs for the alkylrandomization of NP scaffolds. As cell membranes are permeable to Met analogs, this also sets the stage for cell-based and, potentially, in vivo applications. In order to further address the instability of AdoMet and analogs thereof, MAT-catalyzed reactions utilizing Met and ATP isosteres generated highly stable AdoMet isosteres that were capable of downstream utilization by MTs. Finally, the development, use, and results of a high-throughput screen identified mutant-MAT/Met-analog pairs suitable for postliminary bioorthogonal applications.

New antibiotics with novel targets or mechanisms of action are needed to counter the steady emergence of bacterial pathogens that are resistant to antibiotics used in the clinic. MraY, a promising novel target for antibiotic development, initiates the lipid cycle for the biosynthesis of peptidoglycan cell wall, which is essential for the survival of most, if-not-all, bacteria. MraY is an enzyme that catalyzes the transfer and attachment of phospho-MurNAc-pentapeptide to a lipid carrier, undecaprenylphosphate. Muraymycins are recently discovered lipopeptidyl nucleoside antibiotics that exhibit remarkable antibiotic activity against Gram-positive as well as Gram-negative bacteria by inhibiting MraY. We conducted a thorough examination of the metabolic profile of Streptomyces sp. strain NRRL 30473, a known producer of muraymycins. Eight muraymycins were isolated and characterized by a suite of spectroscopic methods, including three new members of muraymycin family named B8, B9 and C5. Muraymycins B8 and B9, which differ from other muraymycins by having an elongated fatty acid side chain, showed potent antibacterial activity against Escherichia coli ∆tolC mutant and pM IC50 against Staphylococcus aureus MraY. Muraymycin C5, which is characterized by an N-acetyl modification of the disaccharide’s primary amine, greatly reduced its antibacterial activity, which possibly indicates this modification is used for self-resistance. In addition to the discovery of new muraymycins, eleven enzymes from the biosynthetic pathway were functionally assigned and characterized in vitro. Six enzymes involved in the biosynthesis of amino ribofuranosylated uronic acid moiety of muraymycin were characterized: Mur16, a non-heme, Fe(II)-dependent α-ketoglutarate: UMP dioxygenase; Mur17, an L-threonine: uridine-5′-aldehyde transaldolase; Mur20, an L-methionine:1-aminotransferase; Mur26, a low specificity pyrimidine nucleoside phosphorylase; Mur18, a primary amine-requiring nucleotidylyltransferase; Mur19, a 5-amino-5-deoxyribosyltransferase. A one-pot enzyme reaction was utilized to produce this disaccharide moiety and its 2′′-deoxy analogue. Two muraymycin-modifying enzymes that confer self-resistance were functionally assigned and characterized: Mur28, a TmrB-like ATP-dependent muraymycin phosphotransferase, and Mur29, a muraymycin nucleotidyltransferase. Notably, Mur28 preferentially phosphorylates the intermediate, aminoribofuranosylated uronic acid, in the muraymycin biosynthetic pathway to produce a cryptic phosphorylated-dissacharide intermediate. Mur23 and Mur24 were assigned as two enzymes that modify the cryptic phosphorylated intermediate by attachment of an aminopropyl group. Mur24 catalyzes the incorporation of butyric acid into the phosphorylated-disaccharide. Following the incorporation, Mur23 catalyzes a PLP-dependent decarboxylation. Finally, Mur15, which belongs to the cupin family, is functionally assigned as a non-heme, Fe(II)-dependent α-ketoglutarate dioxygenase that catalyzes the β-hydroxylation of a leucine moiety in muraymycin D1 to form muraymycin C1. Mur15 can also hydroxylate the γ-position of leucine moiety to muraymycins with fatty acid chain in β-position.

Precision medicine has allowed for the development of monoclonal antibodies that unmask the anti-tumor immune response. These agents have provided some patients durable clinical benefit. However, PD-1 and PD-L1 inhibitor therapies are effective in a small group (10-20%) of non-small cell lung cancer (NSCLC) patients when used as single-agent therapy. The approved companion diagnostic is expression of the immune cell surface molecule, programmed death ligand 1 (PD-L1), on tumors measured by immunohistochemistry (IHC). Studies in tumor biology and immune surveillance dictate that PD-1 inhibitor efficacy should depend on the level of PD-L1 expression; however, the literature has not followed with convincing evidence. The limitations of this test include timing of tissue acquisition, tumor heterogeneity, and timing of therapy relative to the expression of PD-L1. In addition, the requirement of analyzing tumor tissue biopsy samples from a patient is cumbersome. Thus, a peripheral blood biomarker that predicts efficacy of PD-1/PD-L1 inhibition would be optimal for precise and cost-effective treatment. A history of chronic inflammatory diseases may be advantageous for a cancer patient who is treated with PD-1/PD-L1 inhibitors and may allow them to then mobilize a swift immune response to tumor cells. Specific biological components of this persistent inflammation may predict PD-1 inhibitor response. We have taken a novel approach to leverage national healthcare claims data that couples patient history with response to therapy. We have identified potential peripheral blood biomarkers of response to PD-1/PD-L1 inhibitors using a combination of healthcare outcomes and molecular markers that correlate with therapeutic efficacy.

The United States is in the midst of an opioid epidemic. In addition to other system level interventions, almost all states have responded to the crisis by implementing prescription drug monitoring programs (PDMPs). PDMPs are state-level interventions that track the dispensing of Controlled Substances. Data generated at the time of medication dispensing is uploaded to a central data server that may be used to assist in identifying drug diversion, medication misuse, or potentially aberrant prescribing practices. Prior studies assessing the impact of PDMPs on trends in opioid-related morbidity have often failed to take into account the wide heterogeneity of program features and how the effectiveness of these features may be mitigated by insurance status. Previous research has also failed to differentiate the effects of these programs on prescription vs. illicit opioid-related morbidity. The studies in this dissertation attempt to address these gaps using epidemiological techniques to examine the associations between specific PDMP features and trends in prescription and illicit opioid-related poisonings in populations of different insurance beneficiaries. Results of these studies demonstrate that implementation of specific PDMP features is significantly associated with differential trends in prescription and illicit-opioid related poisonings and that the effectiveness of these features vary depending on the insurance status of the population studied. These results suggest that PDMPs offer a valuable tool in addressing the United States’ opioid epidemic, and may be used as empirical evidence to support PDMP best practices in the future.

Acute kidney injury (AKI) is a significant adverse effect of many medications that leads to increased morbidity, cost, and mortality among hospitalized patients. Recent literature supports a strong link between empiric combination antimicrobial therapy and increased AKI risk. As briefly summarized below, the following chapters describe my research conducted in this area. Chapter 1 presents and summarizes the published literature connecting combination antimicrobial therapy with increased AKI incidence. This chapter sets the specific aims I aim to achieve during my dissertation project. Chapter 2 describes a study in which patients receiving vancomycin (VAN) in combination with piperacillin-tazobactam (TZP) or cefepime (CFP). I matched over 1,600 patients receiving both combinations and found a significantly lower incidence of AKI among patient receiving the CFP+VAN combination when controlling for confounders. The conclusion of this study is that VAN+TZP has significantly increased risk of AKI compared to CFP+VAN, confirming the results of previous literature. Chapter 3 presents a study of patients receiving VAN in combination with meropenem (MEM) or TZP. This study included over 10,000 patients and used inverse probability of treatment weighting to conserve data for this population. After controlling for confounders, VAN+TZP was associated with significantly more AKI than VAN+MEM. This study demonstrates that MEM is clinically viable alternative to TZP in empiric antimicrobial therapy. Chapter 4 describes a study in which patients receiving TZP or ampicillin-sulbactam (SAM) with or without VAN were analyzed for AKI incidence. The purpose of this study was to identify whether the addition of a beta-lactamase inhibitor to a beta-lactam increased the risk of AKI. This study included more than 2,400 patients receiving either agent and found that there were no differences in AKI among patients receiving SAM or TZP; however, AKI was significantly more common in the TZP group when stratified by VAN exposure. This study shows that comparisons of TZP to other beta-lactams without beta-lactamase inhibitors are valid. Chapter 5 presents a study of almost 30,000 patients who received combination antimicrobial therapy over an 8-year period. This study demonstrates similar AKI incidence to previous literature and the studies presented in the previous chapters. Additionally, the results of the predictive models suggest that further work in this research area is needed. The studies conducted present a clear message that patients receiving VAN+TZP are at significantly greater risk of AKI than alternative regimens for empiric coverage of infection.

Supersaturating drug delivery systems, such as amorphous solid dispersions (ASDs), have been used extensively to elevate the apparent solubility and oral bioavailability of poorly water-soluble drugs. However, despite the numerous examples of success in increasing solubility and oral bioavailability using ASDs, physical stability challenges remain as formulators seek to employ high drug loading for cost reduction and improved patient compliance. Therefore, stability in both the solid and solution state must be considered for ASDs to be successful. In the solid state, the drug must remain amorphous in the solid matrix throughout the shelf life of the product. Although excipients, such as polymers, have been known to stabilize the amorphous drug in the solid state, stresses encountered during manufacturing and fluctuations in storage conditions may have a detrimental impact on the physical stability of ASDs. Numerous studies have been performed on the impact of each process on ASD stability, yet the relative quantitative impact of each process with respect to the overall energetics landscape is not well understood. Further, ASDs must dissolve after administration and maintain the intended supersaturation in the gastrointestinal (GI) tract during the GI transit time to achieve maximum oral absorption. In solution, the energetics advantage of the amorphous over the crystalline material is a “double-edged sword,” in that it produces not only a high absorption driving force but also an undesirable high crystallization potential. An approach to quantitatively measure the thermodynamic activity of amorphous materials is, thus, desirable. However, it is difficult to measure thermodynamic activity quantitatively, especially due to the speciation process induced by formulation excipients and endogenous materials. Hence, it is often difficult to assess the true enhancement in the absorption for a given ASD and to measure its crystallization tendency in solution. Overall, this dissertation aims to address the following: 1. The relative thermodynamics magnitude of various processes with respect to the crystallization energy associated with amorphous drugs 2. The development of a practical tool to measure the thermodynamic activity of amorphous materials over its crystalline counterpart in solution to assess the enhancement in absorption in the presence of excipients 3. The impact of measured thermodynamic activity on drug crystallization energetics in the presence of excipients

The proteasome is a large protein complex which is responsible for the majority of protein degradation in eukaryotes. Following FDA approval of the first proteasome inhibitor bortezomib for the treatment of multiple myeloma (MM) in 2003, there has been an increasing awareness of the significant therapeutic potential of proteasome inhibitors in the treatment of cancer. As of 2017, three proteasome inhibitors are approved for the treatment of MM but in clinical trials with patients bearing solid tumors these existing proteasome inhibitors have demonstrated poor results. Notably, all three FDA-approved proteasome inhibitors rely on the combination a peptide backbone and reactive electrophilic warhead to target the proteasome, and all three primarily target the catalytic subunits conferring the proteasome’s chymotrypsin-like (CT-L) activity. It is our hypothesis that compounds with non-peptidic structures, non-covalent and reversible modes of action, and unique selectivity profiles against the proteasome’s distinct catalytic subunits could have superior pharmacodynamic and pharmacokinetic properties and may bear improved activity against solid tumors relative to existing proteasome inhibitors. In an effort to discover such compounds we have employed an approach which combines computational drug screening methods with conventional screening and classic medicinal chemistry. Our efforts began with a computational screen performed in the lab of Dr. Chang-Guo Zhan. This virtual screen narrowed a library of over 300,000 drug-like compounds down to under 300 virtual hits which were then screened for proteasome inhibitory activity in an in vitro assay. Despite screening a relatively small pool of compounds, we were able to identify 18 active compounds. The majority of these hits were non-peptide in structure and lacked any hallmarks of covalent inhibition. The further development of one compound, a tri-substituted pyrazole, provided us with a proteasome inhibitor which demonstrated cytotoxic activity in a variety of human solid cancer cell lines as well as significant anti-tumor activity in a prostate cancer mouse xenograft model. We have also evaluated the in vitro pharmacokinetic properties of our lead compound and investigated its ability to evade cross-resistance phenomena in proteasome inhibitor-resistant cell lines. We believe that our lead compound as well as our drug discovery approach itself will be of interest and use to other researchers. We hope that this research effort may aid in the further development of reversible non-peptide proteasome inhibitors and may eventually deliver new therapeutic options for patients with difficult-to-treat solid tumors.

The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6') domain and its mutant enzymes from a bifunctional AME, AAC(6')-Ie/APH(2")-Ia as well as another common AME, APH(3')-IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi.

Background: Azithromycin polarizes macrophages into an alternative phenotype and promotes a regulated immunity. Arginase is an important effector of these macrophages believed to play an essential role in decreasing injury and promoting repair. Hypothesis: Decreases in inflammation in response to Pseudomonas aeruginosa (PA) pneumonia achieved by polarizing macrophages to an alternative phenotype is dependent upon the production of arginase. Methods: Requirement of arginase was examined by pharmacological inhibition using S-(2-boronoethyl)- l-cysteine (BEC) or l-norvaline and by infecting arginase-1 conditional knock-out mice (Arg1flox/flox;Lyz2-cre (Arg1Δm)) with PA intratracheally. Arg1ΔM and control Arg1flox/flox mice were then dosed with azithromycin daily via oral gavage beginning four days prior to infection. Analysis of weight loss in addition to characterization of inflammatory cells and cytokine production via flow cytometry was performed. Macrophages were then stimulated with LPS and polarized with IL4/13, IFNγ, or azithromycin plus IFNγ. Western blot for signaling mediators, p65 translocation assay, and immunofluorescence were performed. Results: Myeloid arginase-1 deletion resulted in greater morbidity along with more severe inflammatory response compared to the Arg1flox/flox mice. Arg1Δm mice had greater numbers of neutrophils, macrophages, and lymphocytes in their airways and lymph nodes compared to the Arg1flox/flox mice. Conversely, global arginase inhibition resulted in greater weight loss along with greater neutrophil and macrophage infiltration compared to Arg1Δm mice. BEC and l-norvaline treated mice had higher numbers of lymphocytes in their lymph nodes with variable effects on airway lymphocyte counts. Azithromycin treatment comparably reduced the acute inflammatory responses in both Arg1Δm and Arg1flox/flox mice. To evaluate this mechanism, we show in vitro that azithromycin decreases NF-κB activation by preventing p65 nuclear translocation and by decreasing STAT1 activation in a concentration-dependent manner. These effects were reversed with IKKβ inhibition. Conclusions: Myeloid arginase is essential for control of inflammatory responses in PA pneumonia with potentially different effects of other cellular sources demonstrated with global arginase inhibition. Azithromycin reduces excessive inflammation even in the absence of arginase, potentially through a cross-inhibitory mechanism involving STAT1 and NF-κB pathways through IKKβ.