Ever since the advent of computer-aided drug design (CADD), in silico simulation methods have greatly accelerated the drug discovery process and lead to the discovery of numerous drug candidates. With the exponential growth of computational power, we nowadays simulate biologic systems at a scale unimaginable a decade ago and thus provides perspectives for drug design. In this dissertation research, combining in silico simulation methods like molecular docking and molecular dynamics (MD) simulation with organic synthesis, in vitro/in vivo experiments and clinical data mining, we developed new drug discovery strategies. These strategies were applied in our drug discovery projects and led to the discovery of inhibitors of microsomal prostaglandin E2 synthase 1 (mPGES-1) and butyrylcholinesterase (BChE) as potential drug candidates. Protein mPGES-1 is known as an ideal target for next generation of anti-inflammatory drugs without the side-effects of currently available anti-inflammatory drugs. Unfortunately, almost all the previously reported human mPGES-1 inhibitors are inactive (or possess very low activity) against mouse or rat mPGES-1 that prevents using well-established mouse/rat models of inflammation, pain, and other diseases for preclinical studies. It would be extremely challenging for the mPGES-1-based drug development to follow traditional drug discovery and development route. In order to solve this problem, we developed and applied Drug Repurposing Effort Applying Integrated Modeling-in vitro/vivo-Clinical Data Mining (DREAM-in-CDM) strategy in this project. With molecular dynamics simulation, we observed the process of how mPGES-1 adopts an alternative conformation to control the access of co-factor GSH (glutathione) and its impact on the function of the protein. Based on the simulation results, we not only found an explanation for the difference between the X-ray and CryoEM (cryogenic electron microscopy) structure of mPGES-1 but also used molecular docking method to identify FDA approved drug, lapatinib, as an mPGES-1 inhibitor by virtual screening and the subsequent in vitro experiments. By mining the available clinical trial data, we found solid evidence that lapatinib can be used to relieve various types of pain in cancer patients. Since lapatinib is very well tolerated, we expect lapatinib to be repurposed as a new treatment for cancer-related pain. BChE has been identified as an ideal drug target for the treatment of Alzheimer’s disease (AD) and heroin overdose. The selectivity of a therapeutically useful inhibitor for BChE over AChE is very important. Unfortunately, there is no good selective BChE inhibitor. With a robust and virtual screening strategy combining with in vitro experiments, we identified a series of compounds from the NCI compound depository as BChE inhibitors with novel scaffolds, high activity and selectivity at the same time. The most potent compound was re-synthesized and the enantiomers of the compound were separated for the first time. The binding mode of the most potent compound was also analyzed and the origin of its high activity and selectivity was revealed that will guide the development of BChE selective inhibitors in the future. In addition, a new tacrine-based BChE affinity chromatography resin was developed. The developed new resin has enabled us to more conveniently and efficiently purify the BChE proteins with improved high purity. In general, we have successfully developed new drug discovery strategies to identify novel inhibitors of different enzymes. With these newly developed strategies, we expect additional drug discoveries to be made in the foreseeable future.

Pain is an unpleasant sensory and emotional experience associated with actual or potential tissus damage or described in terms of such damage. Opioids are effective in treating moderate to severe pain, but opioid alone therapy is associated with several adverse effects, development of tolerance and addiction potential. One way to solve these problems is to administer opioids with adjuvant drugs. In this project several opioid molecules were combined with other adjuvant drugs in a single chemical entity to form a codrug. A series of codrugs were prepared by conjugation of an opioid with S-(-)-nornicotine, ketamine, norketamine and gabapentin. Several of the synthesized codrugs were evaluated for analgesic activity in the rats after oral administration. Codeine-S-(-)- nornicotine, 3-O-acetylmorphine-S-(-)-nornicotine, and N-ethoxycarbonylgabapentincodeine codrugs showed greater effectiveness as well as prolonged pain management properties as compared to the parent drugs. Stabilities of several synthesized codrugs were studied in aqueous solutions from pH 1.3-7.4, in simulated gastrointestinal fluids, in rat plasma and in brain homogenate. Only the ester-linked codrugs showed sign of hydrolysis in different solutions. Carbamate-linked codrugs didn’t cleave under any hydrolytic condition. Pharmacokinetic study was performed on the following three codrugs: 3-O-acetylmorphine-S-(-)-nornicotine, N-acetylgabapentin-codeine, and N-ethoxycarbonylgabapentin- codeine. The carbamate linkage in 3-O-acetylmorphine-S-(-)- nornicotine codrug did not cleave in vivo to produce parent drugs. The ester linkage in N-acetylgabapentin- codeine codrug cleaved in vivo to produce codeine and N-acetylgabapentin, but N-acetylgabapentin did not undergo hydrolysis to produce gabapentin. The ester linkage in N-ethoxycarbonylgabapentin-codeine codrug hydrolyzed slowly in plasma to produce N-ethoxycarbonylgabapentin and codeine and then the carbamate linkage in N-ethoxycarbonylgabapentin hydrolyzed even slowly to produce gabapentin. Produced codeine also metabolized to generate some amount of morphine. Thus, the design and synthesis of an opiate and gabapentin codrug was achieved which was stable enough in the gastrointestinal tract, showed enhanced analgesic effects as compared to the physical mixture of the parent drugs, and also produced the two parent drugs in blood plasma.

Over a decade, proteasome inhibitors (PIs), bortezomib, carfilzomib (Cfz) and ixazomib, have contributed to a significant improvement in the overall survival for multiple myeloma (MM) patients. However, the response rate of PI was fairly low, leaving a huge gap in MM patient care. Given this, mechanistic understanding of PI resistance is crucial towards developing new therapeutic strategies for refractory/relapsed MM patients. In this dissertation work, we found H727 human bronchial carcinoid cells are inherently resistant to Cfz, yet susceptible to other PIs and inhibitors targeting upstream components of the ubiquitin-proteasome system (UPS). It indicated H727 cells may serve as a cell line model for de novo Cfz resistance and remains UPS dependent for survival. To examine the potential link between proteasome catalytic subunit composition and cellular response to Cfz, we altered the composition of proteasome catalytic subunits via interferon-γ treatment or siRNA knockdown in H727 cells. Our results showed alteration in composition of proteasome catalytic subunits results in sensitization of H727 cells to Cfz. It supported that proteasome inhibition by alternative PIs may still be a valid therapeutic strategy for patients with relapsed MM after having received treatment with Cfz. With this in mind, we designed and synthesized a small library of epoxyketone-based PIs by structural modifications at the P1′ site. We observed that a Cfz analog, harboring a hydroxyl substituent at its P1′ position was cytotoxic against cancer cell lines with de novo or acquired resistance to Cfz. These results suggested that peptide epoxyketones incorporating P1′-targeting moieties may have the potential to overcome Cfz resistance mechanisms in cells. The immunoproteasome (IP), an inducible proteasome variant which is harboring distinct catalytic subunits, LMP2, MECL1 and LMP7 of the proteasome typically expressed in cells of hematopoietic origin, plays a role in immune response and is closely linked to inflammatory diseases. It has been reported that the IP is upregulated in reactive glial cells surrounding amyloid β (Aβ) deposits in brains of Alzheimer’s disease (AD) patients and AD animal models. To investigate whether the IP is involved in the pathogenesis of AD, we examined the impact of IP inhibition on cognitive function in AD mouse models. We observed that YU102, an epoxyketone peptide targeting the IP catalytic subunit LMP2, improved cognitive dysfunction in AD mice without clearance of Aβ deposition or tau aggregation. Our cell line model study also showed a potential mode of action of YU102 which is suppressing pro-inflammatory cytokine production in microglial cells. It suggested that LMP2 contributes to microglia-mediated inflammatory response. These findings supported that LMP2 may offers a valuable therapeutic target for treatment of Alzheimer’s disease, expanding the therapeutic potential of the LMP2-targeting strategy.

In spite of great effort aimed at eradication, the malaria epidemic still claims over 600,000 lives each year, and 50% of the world is at risk of contracting the disease. The most deadly form of malaria is caused by Plasmodium falciparum, which is spread from human to human via the female Anopheles mosquito. P. falciparum's lifecycle, which includes both sexual and asexual reproduction, facilitates rapid evolution in response to drug pressure, resulting in the emergence of resistant strains against every antimalarial medication that has been deployed. There is a great need for new antimalarial drugs. Chloroquine (CQ), an aminoquinoline drug deployed in the 1940s, was an inexpensive, effective and safe drug but now has been rendered ineffective throughout much of the tropical regions due to the emergence of CQ-resistant strains of P. falciparum. A new class of hybrid drugs, called Reversed-CQs, has been developed by linking a molecule with a CQ-like moiety to a molecule with a reversal agent (RA) moiety; an RA is a chemosensitizer that can reverse CQ-resistance. The prototype Reversed-CQ, PL01, was shown to be effective in vitro against sensitive and resistant P. falciparum cell cultures, with IC50 values of 2.9 and 5.3 nM, respectively, in comparison to IC50 values for CQ which were 6.9 and 102 nM, respectively. In the course of the Reversed-CQ research, PL74 was synthesized with a pyridine ring replacing the quinoline ring. It was expected that PL74 would display reversal agent activity but would not display antimalarial activity. However PL74 showed antimalarialactivity with IC50 values of 185 and 169 nM in vitro against CQ-sensitive and CQ-resistant strains, respectively. In the investigation of PL74 it has been found that this molecule has a pyridinium salt structure, novel to the Reversed-CQ compounds, and through a structure-activity relationship (SAR) study, it was shown to have activity that may indicate a mode of action different from the Reversed-CQ compounds. A study of the literature revealed that pyridinium salt compounds, with some similarity to PL74, were found to operate as choline analogs inhibiting the biosynthesis of phosphatidylcholine as their main antimalarial mode of action.

For cardiovascular diseases such as high blood pressure, angina pectoris, and left ventricle hypertrophy; long-term activation of beta-adrenergic receptors is strongly linked to the progression of these diseases. A class of antagonistic drugs that target betaadrenergic receptors are collectively called beta-blockers. These drugs are commonly used to reduce the inotropic and chronotropic effects of beta-adrenergic receptor activation. This past decade has revealed that beta-blockers and other ligands are capable of functional selectivity at receptors. Functional selectivity describes the ability of ligands acting at 0 protein-coupled receptors (OPCRs) to preferentially activate or inhibit different signal transduction pathways. The studies on beta-adrenergic 2 receptors that explored functional selectivity showed that beta-blockers can be functionally selective by inhibiting the cAMP pathway while simultaneously activating ERK. The 0 protein coupled to beta-adrenergic receptors are the primary regulators of the cAMP, however there are a variety of pathways that can regulate ERK activity and few studies have tried to determine which pathway(s) the beta-blockers are targeting to cause this ERK activation. This is especially important for beta-adrenergic 2 receptors because they can activate ERK through multiple pathways (0 protein switching from G, to Oi/oprotein, beta-arrestin assisted or EOFR transactivation). ERK activation is linked to reversing cell damage caused by apoptosis signaling that results from G, activation by beta-adrenergic receptors. Understanding the specific pathways these beta-blockers can target for ERK activation would lead to better understanding of their therapeutic benefits. In this study we plan to elucidate the pathways several beta-blockers are targeting to activate ERK. In particular, we will investigate the role of clathrin-mediated receptor internalization and EGFR transactivation in beta-blocker-dependent ERK phosphorylation. In HEK 293 cells transfected with beta-adrenergic 2 receptors, we measured the changes in cAMP and ERK phosphorylation in response to the following beta-blockers labetalol, alprenolol, bucindolol, carvedilol, carazolol, leI 118,551 and propanolol. All of the beta-blockers studied inhibited isoproterenol-stimulated cAMP accumulation but stimulated the phosphorylation of ERK to varying degrees. Beta-blocker-mediated ERK phosphorylation was shown to be dependent on clathrin-dependent internalization and EGFR transactivation.

Commercial pharmacokinetic programs are used to provide clinicians with the tools to predict pharmacokinetic models, estimate pharmacokinetic parameters, and analyze serum data for efficient and consistent drug therapy and, therefore, help clinicians to optimize drug therapy. Many of the commercial programs use different methods of data entry and analysis. In this study, three commercial dosing programs, Kinetidex®, DataKinetics®, and Simkin®, were utilized to evaluate their performance on predicting gentamicin initial dosage regimens and adjusted dosage regimens. The performance of the three programs were compared by measuring the difference in the percentage of the prediction error (PE) as bias and absolute prediction error (APE) as precision using a modified method developed by Sheiner. A clinically significant difference in outcomes was determined to exist if the difference in the calculation of the dosage regimen obtained from the programs as compared to a reference calculated dosage regimen exceeded 1 0 %. A statistically significant difference in the calculation of the dosage regimens obtained from the programs was determined by ANOVA testing (p < 0.05). The results indicated that the Simkin® program had the tendency to overestimate loading doses, and the difference as compared to the reference data was clinically significant. Also the difference observed in calculating loading doses between the Simkin® program and the other two programs was statistically significant. The Kinetidex® program had bias by underestimating daily maintenance doses, and the difference as compared to the reference data was clinically significant. The difference as compared to the reference data for calculating the daily maintenance doses by the DataKinetics® and Simkin® program did not exhibit bias, but the difference was clinically significant. The difference in the performance of predicting daily maintenance doses by the programs was statistically significant in bias but not in precision. The results of computer predicted serum levels versus the measured serum levels indicated that the Kinetidex® program exhibited bias by overestimating the peak and trough concentrations as compared to the other two programs, but there is no statistically significant difference among the programs in precision. In calculating adjusted doses using the measured serum levels, the results obtained from the three programs showed no difference in bias or precision. A comparison of the results of computer-predicted serum levels showed no difference among the programs in bias and precision. In conclusion, the study showed that there was a difference in the predicting ability of the three programs in calculating initial dosage regimens but showed no statistical difference in calculating the total daily-adjusted doses when using patient serum levels.

Transthyretin (TTR) amyloidosis is a progressive, fatal disease in which deposition of amyloid derived from either mutant or wild-type TTR causes severe organ damage and dysfunction. TTR cardiomyopathy is an infiltrative, restrictive cardiomyopathy characterized by progressive left and right heart failure. Familial amyloid cardiomyopathy (FAC) is driven by pathogenic point mutations in the TTR gene that destabilize the TTR tetramer, prompting its dissociation into dimers and monomers, with subsequent misfolding, aggregation and deposition of toxic TTR amyloid aggregates in the myocardium. The most prevalent mutation that causes FAC is the V122I variant, carried by 3.4% of African Americans, that increases the risk of cardiomyopathic events several-fold in this population. AG10, a potent TTR kinetic stabilizer, prevents dissociation of V122I-TTR in serum samples obtained from patients with FAC. Further, we have described structural, biochemical, and animal studies of AG10 which reveal mechanistic and structural insights on the ability of AG10 to mimic the disease suppressing T119M variant in stabilizing TTR. The second part of the thesis discusses harnessing TTR as a platform to enhance in vivo half-life (t1/2) of therapeutic peptides. Native peptides typically display short in vivo t1/2, however conjugation of peptides to macromolecules causes steric hindrance which often harms the binding of peptides to target receptors, compromising the in vivo efficacy. Utilizing Gonadotropin Releasing Hormone (GnRH) as a model peptide, we show that t1/2 may be extended without compromising potency. Our approach involves endowing peptides with a small molecule that binds reversibly to the serum protein transthyretin. Our strategy was effective in enhancing the t1/2 of an agonist for GnRH receptor while maintaining its binding affinity, which was translated into superior in vivo efficacy. The third and final part of the thesis describes our effort on developing a fluorescent probe to quantify TTR in human serum using fluorescence polarization. TTR is used as a marker for nutritional and inflammatory status in critical patients. This assay development has the potential to minimize lab cost, effort, and time with regards to determination of TTR concentration in patients.

Gilvocarcin V (GV) belongs to the angucycline class of antibiotics that possesses remarkable anticancer and antibacterial activities with low toxicity. Gilvocarcin exhibits its light induced anticancer activity by mediating crosslinking between DNA and histone H3. When photo-activated by near-UV light, the C8 vinyl group forms a [2+2] cycloadduct with thymine residues of double stranded DNA. D-fucofuranose is considered essential for histone H3 interactions. However, the poor water solubility has rendered it difficult to develop gilvocarcin as a drug. We aim to design novel gilvocarcin analogues with improved pharmaceutical properties through chemo-enzymatic synthesis and mutasynthesis. Previous studies have characterized many biosynthetic genes encoding the gilvocarcin biosynthetic skeleton. Despite these previous findings the exact functions of many other key genes are yet to be fully understood. Prior gene inactivation and cross-feeding experiments have revealed that the first isolable tetracyclic aromatic product undergoes a series of steps involving C–C bond cleavage followed by two O-methylations, a penultimate C-glycosylation and final lactone formation in order to fully develop the gilvocarcin structure. To provide a deeper understanding of these complex biochemical transformations, three specific aims were devised: 1) synthesis of the proposed intermediate and in vitro enzyme reactions revealed GilMT and GilM’s roles in gilvocaric biosynthesis; 2) utilizing in vitro studies the enzyme responsible for the C–C bond cleavage and its substrate were determined; 3) a small series of structural analogues of the intermediate from the gilvocarcin pathway was generated via chemical synthesis and fed to the mixture of the enzymes, GilMT and GilM. These reaction mixtures were then analyzed to establish the diversity of substrates tolerated by the enzymes.

Today, more Americans die each year because of drug overdoses than are killed in motor vehicle accidents. In fact, in 2015, more than 33,000 individuals died due to an overdose of heroin or prescription opioids. Sadly, 40-60 % of patients on current opioid addiction treatment medications relapse. Studies have shown that the addiction/abuse liability of opioids are abolished in mu opioid receptor (MOR) knock-out mice; this indicates that the addiction and abuse liability of opioids are mainly mediated through MOR. Utilizing the “message-address concept”, the our laboratory reported a novel non-peptide, reversible MOR selective ligand 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α (isoquinoline-3-carboxamido)morphinan (NAQ). Molecular modeling and mutagenesis studies revealed that the selectivity of NAQ for MOR is because of the π-π stacking of the isoquinoline ring of NAQ with W318. Therefore, other heterocyclic ring systems were explored to obtain a diverse library of compounds with similar or different molecular interactions and pharmacologic characteristics as NAQ. The newly designed compounds were indole analogs of 6α/β-naltrexamine. The compounds were synthesized and the affinity and selectivity for MOR determined using the radioligand binding assay while the functional activity at MOR was determined using the [35S]GTPγS binding assay. The indole analog of 6α-naltrexamine substituted at position 7 (compound 6) was found to be very potent and had the lowest efficacy in the [35S]GTPγS functional assay while the indole analog of 6β-naltrexamine substituted at position 2 (compound 10) was identified as a MOR agonist and had the greatest efficacy. In vivo studies were conducted using the warm-water immersion assay to find whether the synthesized compounds had antinociceptive effects and/or blocked the antinociceptive effects of morphine. Not surprisingly, compound 10 was identified as an opioid agonist while compound 6 almost completely blocked morphine’s antinociceptive effects. The opioid antagonist effect of compound 6 was found to be dose dependent with an AD50 of 2.39 mg/kg (0.46-12.47). An opioid withdrawal assay was conducted on compound 6 using morphine-pelleted mice. Compound 6 produced significantly less withdrawal symptoms at 50 mg/kg than naltrexone at 1 mg/kg. Therefore, compound 6 has the potential to be used in opioid addiction and withdrawal treatment.

While protein α-N-terminal methylation has been known for nearly four decades since it was first uncovered on bacteria ribosomal proteins L33, the function of this modification is still not entirely understood. Recent discoveries have demonstrated α-N-terminal methylation is essential to stabilize the interactions between regulator of chromosome condensation 1 (RCC1) and chromatin during mitosis, to localize and enhance the interaction of centromere proteins (CENPs) with chromatin, and to facilitate the recruitment of DNA damage-binding protein 2 (DDB2) to DNA damage foci. Identification of N-terminal methyltransferase 1 (NTMT1) unveiled the eukaryotic methylation writer for protein α-N-termini. In addition, NTMT2 that shares over 50% sequence similarity, has been identified as another mammalian protein α-N-terminal methylation writer. Knockdown of NTMT1 results in mitotic defects and sensitizes chemotherapeutic agents in breast cancer cell lines, while NTMT1 knockout mice showed premature aging. Additionally, NTMT1 has been shown to be overexpressed in a colorectal and melanoma tumor tissues, and in lung and liver cancer cell lines. Given the vast array of clinical relevance, chemical probes and inhibitors for NTMT1 are vital to elucidate information about the function and downstream process of protein α-N-terminal methylation. Therefore, 47 peptidomimetic compounds have been synthesized that target NTMT1. These peptide-based compounds range from three to six amino acids in length and the top 5 compounds have 3- to 300- fold selectivity for NTMT1 compared to other methyltransferases. An inhibition mechanism study has also been performed to verify the inhibitors are targeting the NTMT1 peptide binding site. Seven compounds have an IC50 of less than 5 µM and our top inhibitor, BM-47, has an IC50 of 0.32 µM ± 0.06 for NTMT1. To further elucidate information about the NTMTs and their downstream effects, we utilized photoaffinity probes to target these enzymes. Our 6 photoaffinity probes exhibited in a dose- and time-dependent manner. Probe labeling has been shown to be driven by recognition and selectively and competitively label the NTMT writers in a complex cellular mixture. Our results also provided the first indication of substrate preferences among NTMT1/2. Methylated photoaffinity probes were also synthesized to identify novel proteins that recognize a methylated N-terminus and shed light on the function of α-N-terminal methylation.