This research developed and validated QSPKR models for predicting in-vivo human, systemic biologically relevant PK properties (i.e., reflecting the disposition of the unbound drug) of four, preselected, pharmacological classes of drugs, namely, benzodiazepines (BZD), neuromuscular blocking agents (NMB), triptans (TRP) and class III antiarrhythmic agents (AAR), as well as PK allometric scaling (PK-AS) models for BZD and NMB, using pertinent human and animal systemic PK information (fu, CLtot, Vdss and fe) from published literature. Overall, lipophilicity (logD7.4) and molecular weight (MW) were found to be the most important and statistically significant molecular properties, affecting biologically relevant systemic PK properties, and the observed relationships were mechanistically plausible: For relatively small MW and lipophilic molecules, (e.g., BZD), an increase in logD7.4 was associated with a decrease in fu, an increase in Vdssu and CLnonrenu, suggesting the prevalence of nonspecific hydrophobic interactions with biological membranes/plasma proteins as well as hepatic partitioning/DME binding. Similar trends were observed in fu and Vdssu for intermediate to large MW, hydrophilic molecules (e.g., NMB). However, although similar trends were observed in fu and Vdssu for relatively hydrophilic, intermediate MW molecules (e.g., TRP), and a heterogeneous class (e.g., Class III AAR), logD7.4 and MW were found to be highly correlated, i.e., the indepdendent effects of logD7,4 and MW cannot be assessed NMB, TRP and Class III AAR show mechanistically diverse clearance pathways, e.g., hepatobiliary, extrahepatic, enzymatic/chemical degradation and renal excretion; therefore, effects of the logD7.4 and/or MW are note generalizable for any of the clearances across classes. PK-AS analyses showed that Vdssu and Vdss scaled well with body weight across animal species (including humans) for BZD. Overall, within the limitations of the methods (and the sample size), ‘acceptable’ predictions (i.e., within 0.5- to 2.0-fold error range) were obtained for Vdssu and Vdss for BZD (and fu correction resulted in improvement of the prediction); however, none of the CLtot predictions were acceptable, suggesting major, qualitative interspecies differences in drug metabolism, even after correcting for body weight (BW). NMB undergo little extravascular distribution owing to their relatively large MW and charged nature, and, as a result, a high percentage of acceptable predictions was obtained for Vdss (based on BW). Similarly, the prediction of CLren (based on BW and glomerular filtration rate, GFR) was acceptable, suggesting that NMB are cleared by GFR across species, and there are no interspecies differences in their tubular handling. On the other hand, CLtot (and/or CLnonren) could not be acceptably predicted by PK-AS, suggesting major differences in their clearance mechanisms across animal species.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by beta-amyloid (Aβ) aggregation/oligomerization, biometal dyshomeostasis, oxidative stress, and neuroinflammation. The multifactorial nature of AD may indicate the therapeutic potential of multifunctional ligands that tackle various risk factors simultaneously as effective AD-modifying agents. This notion is further supported by the fact that while numerous AD-modifying agents targeting one single risk factor have been developed and a number of them entered clinical trials, none of them has been successfully approved by the FDA. Furthermore, neuronal cell membrane/lipid rafts (CM/LR) have been demonstrated to associate with all the indicated risk factors, indicating that this relationship can be exploited therapeutically to design strategically distinct multifunctional ligands by incorporating CM/LR anchorage into molecular design. With the long-term goal of developing multifunctional ligands to slow or stop the progression of AD, recently we have embarked on the development of bivalent multifunctional Aβ oligomerization inhibitors (BMOIs) as potential AD-modifying agents. These BMAOIs contain curcumin as the multifunctional moiety and cholesterol as the CM/LR anchorage moiety linked by a spacer to co-target AβOs, CM/LR, and oxidative stress. The hypothesis of the BMAOI strategy is that BMAOIs will anchor/target the multifunctional AβO inhibitor moiety inside, or in the vicinity of, CM/LR in which Aβ oligomerization, Aβ/biometal interaction and oxidative stress occur to efficiently interfere with these processes. In support of this hypothesis, proof-of-concept of the BMAOIs strategy has been reached through our preliminary studies. Our results demonstrated that: 1) BMAOIs containing curcumin as the multifunctional AβO inhibitor and cholesterol as CM/LR anchor primarily localize to CM/LR while curcumin does not; 2) BMAOIs with optimal spacer length efficiently inhibit the production of intracellular AβOs and protect MC65 cells from AβO-induced cell death (EC50~3 µM) while curcumin exhibits no significant activity; 3) these active BMAOIs retain curcumin’s antioxidant and metal complexation properties. Our preliminary studies also demonstrated the critical roles of spacer length and connectivity in the molecular design of BMAOIs and one lead compound was identified for further structural modification and optimization. Furthermore, this lead compound was shown to cross the blood-brain barrier (BBB) in a preliminary in vivo study as well as bind to Aβ plaques. Taken together, these results clearly reach the proof-of-concept of BMAOIs and confirm the rationale of designing BMAOIs to develop potential AD-modifying agents. In this thesis, we continued the exploration and validation of the BMAOI strategy by designing and biological characterizing a series of BMAOIs containing cholesterylamine as the CM/LR anchorage moiety and curcumin as the multifunctional moiety. Ten BMAOIs with the spacer length of 15, 17, 19, 21, and 23 atoms were designed and synthesized. Initially, these BMAOIs were tested for the neuroprotective activity against the AβO-induced cytotoxicity in human neuroblastoma MC65 cells. Then, Western blot analysis was performed for active BMAOIs to confirm the association of neuroprotection and suppression of AβOs. Furthermore, active BMAOIs were examined for antioxidant and metal complexation properties. Finally, Aβ plaque binding was examined using transgenic AD mice brain sections. Our results demonstrated that the same spacer length but different connectivity are preferred in this new series of BMAOIs for neuroprotective activity as that of the lead compound from cholesterol series. Moreover, the neuroprotection activity is closely associated with the inhibition of AβOs as demonstrated by Western blot analysis. In addition, the active BMAOIs retain the antioxidant and biometal binding properties of curcumin. More importantly, the binding affinity to the Aβ plaques was again confirmed for the new BMAOIs containing cholesterylamine. In summary, the design and characterization of the new series BMAOIs further confirmed the rationale of BMAOI strategy and their potential to lead to a new direction in development of effective AD-modifying and treatment agents.

The chemokine receptor CCR5 has been implicated in the pathogenesis of cancers and AIDS. A series of novel piperidine derivatives were designed, synthesized, and evaluated as CCR5 antagonists. The ability of the new ligands to inhibit the increment of intracellular calcium level stimulated by endogenous ligand CCL5 was measured in the calcium mobilization assay as an indication of its CCR5 receptor antagonism. The anti-proliferation assay was performed to measure the ability of these new compounds to inhibit the proliferation of prostate cancer cell lines, PC-3 and M12. A new lead compound has been identified which showed micromolar level of inhibition to PC-3 cell line proliferation as well as calcium mobilization. These studies are the beginning of a thorough analysis of the CCR5 receptor antagonist binding pocket in the CCR5 receptor. Further examination may help identify next generation lead to develop highly selective CCR5 receptor antagonists and anti prostate cancer agents.

Current anticoagulants carry a serious risk of bleeding complications. In addition, narrow therapeutic index, drug interactions, immunological reactions, toxicity and high cost to benefit ratio limits the effective use of these drugs in patients with thrombotic conditions.Heparin is the most widely used anticoagulant. We hypothesized that one of the major drawback of heparins, its non-specific interaction with the plasma proteins arises as a result of negative charges. To reduce these non-specific interactions, our laboratory designed sulfated low molecular weight lignin (LMWL) like biomacromolecules, which were found to be direct inhibitors of thrombin and factor Xa, acting through a unique exosite-2 mediated process. To elucidate the structural basis of this mechanism, we studied unsulfated and size fractionated LMWLs. Detailed enzyme inhibition studies with sulfated and unsulfated LMWLs of ferulic and caffeic acid oligomers revealed that sulfation was not absolutely critical for dual inhibition property and smaller oligomers can yield a potent anticoagulant. Mechanistically, unsulfated LMWLs retained exosite-2 mediated inhibition mechanism. A major advantage expected of the unsulfated LMWLs is the possibility that orally bioavailable anticoagulants may become possible.To identify target specific structures within the heterogeneous population of sulfated LMWLs, we prepared sulfated β-O-4-linked oligomer using chemical synthesis. Enzyme inhibition studies revealed that the sulfated β-O-4 LMWL were highly selective direct inhibitors of thrombin. These results show for the first time that specific structural features on LMWL scaffold dictate inhibition specificity. Studies in plasma and blood display highly promising anticoagulant profile for further studies in animals. To further study the LMWL scaffold as macromolecular mimetic of heparin; we investigated their effect in preventing cellular infection by herpes simplex virus-1 (HSV-1). Based on previous findings on sulfated lignins a size-dependent study on unsulfated LMWLs was done. The unsulfated lignins were found to not only inhibit HSV-1 entry into mammalian cells, but were more potent than sulfated lignins. Interestingly, shorter chains were found to be as active as the longer ones, suggesting that structural features, in addition to carboxylate groups, may be important. It can be expected that unsulfated lignins also antagonize the entry of other enveloped viruses, like HIV-1 and HCV that utilize heparan sulfate to gain entry into cells. The results further present major opportunities for developing lignin-based antiviral formulations for topical use.

Biophysical entities are complex systems systems with strong environmental coupling, dominated by fluctuations on a hierarchy of timescales. These properties confound simulation of ligand binding and catalysis, inflating the scale of the problem to one tractable only with a considerable outlay of resources. In an attempt to ameliorate this restriction, several techniques are developed to accelerate biomolecular simulations while collaterally lending physical insight. The first segment of this dissertation is concerned with directed simulations of ligand binding in a model system. Using the serum retinol binding protein as a prototype, the potential of mean force associated with ligand binding is calculated and dissected. Desolvation is sufficient to drive formation of an intermediate binding state; however, a combination of electrostatic and van der Waals interactions pull the intermediate into a stable configuration. Association is accompanied by a change in the conformational flexibility of the portal domains of sRBP and subsequent "stiffening" of the holo sRBP, reflecting an "order-disorder" transition in the protein. The third and fourth chapters of this dissertation entail ab initio molecular dynamics (AIMD) and quantum Monte Carlo methods (QMC) for computational enzymology. An ideal system for the application of AIMD, are the cytochromes P450 (CYP450s). Most AIMD calculations are performed using plane-wave (PW) density functional theory as an electronic structure method; conversely, computational enzymology is generally performed using calculations with Gaussian basis sets. In this scenario, no benchmark exists to comparison of PW calculations with experimental data. To clarify this situation, benchmark PW calculations are performed on CYP450 Compound I, the iron-oxo species operant in these enzymes. Finally, lattice QMC methods are developed to characterize tunneling in mean-field backgrounds. Using AIMD simulations, a potential of mean force is constructed in the limit of classical nuclei. A framework for path integral Monte Carlo is introduced in which the Euclidean functional integral is discretized on a lattice, permitting calculations of correlation functions and ultimately the action of the system. As the action is quenched, instanton solutions and their contribution to degeneracy splitting are obtained. This technique is demonstrated for malonaldehyde, a system in which proton tunneling is critical.

Heparin is a natural allosteric modulator, with numerous structural and conformational variations leading to many reports of bleeding complications and variations in anticoagulant effects. A flurry of research has been directed towards understanding this puzzle. This work entails the utilization of three unique strategies to further our understanding of this complex issue. Traditional synthetic, biosynthetic and biophysical approaches have failed to conquer the GAG-protein complexity. Computational analysis however could serve as a powerful approach to decipher this dilemma. A dual filter algorithm was incorporated to identify unique hexasaccharide sequences for HCII and AT. Our experimental studies exhibit a good correlation with our computational findings in addition, to the discovery of the first reported heparin based hexasaccharide sequence (HX1) as a potent activator of HCII and AT. In contrast to the enormity of GAG sequences, there appears to be a pattern where rare sequences have been identified to modulate characteristic functions in proteins. Our search led us to a biosynthetically rare GAG residue 2-O-sulfated glucuronic acid (GlcAp2S). Our computational studies indicated elements of selective recognition with coagulation enzymes propelling us towards synthesizing a polymer, HS2S2S enriched in GlcAp2S and GlcNp2S saccharides. Our biological studies indicate its potential in activating AT and HCII in addition to a previously unobserved inhibition of thrombin but not FXa, which is corroborated by our computational studies. These studies therefore showcase the importance of studying rare sequences to further our understanding of differential recognition of proteins of the coagulation cascade. An alternate anticoagulant strategy involves utilization of upstream enzymes like FXIa. Consequently, we devised a rational strategy, which targets the differential hydrophobic domain near the heparin binding sites of proteins through the design of molecules termed as sulfated allosteric modulators. Our endeavor led to the discovery of a library of quinazolin4-(3H)ones) dimers as selective inhibitors of FXIa. We recognized the linker length and geometry to be an important element affecting potency and selectivity. We therefore synthesized a library of 18 dimers using simple reaction schemes. Our inhibition studies do highlight a 9-fold improvement in potency.

Objective: To determine if the prevalence of anti-diabetic and antilipidemic medication use among children treated with atypical antipsychotics is higher than those not treated with antipsychotics. Methods: Virginia Medicaid beneficiaries (2-17 years) continuously enrolled from August 1, 2010 to July 31, 2011 with at least two prescription claims for atypical antipsychotics were the exposed group. All other subjects during the study period were the non-exposed group. Prevalence of anti-diabetic and antilipidemic medication use in both groups were computed and compared using Chi-square test (α=0.05). Results: A total of 299,593 and 4,922 subjects were identified as the non-exposed and exposed groups, respectively. Prevalence of anti-diabetic medication use was 0.32% in the unexposed and 1.40% in the exposed group (p<0.0001). Prevalence of antilipidemic medication use was 0.09% in the unexposed and 0.35% in the exposed group (p<0.0001). Conclusion: Prevalence of anti-diabetic and antilipidemic medication use in the exposed group was significantly higher.

With over 140 vitamin B6 (Pyridoxal 5’-phosphate, PLP) dependent enzymes, serving vital roles in various transamination, decarboxylation, retro-aldol cleavage and synthesis pathways these enzymes constitute the most versatile catalytic systems in nature. Enzymes of this group have an inherent reaction as well as substrate specificity. A single co-factor namely, PLP is used by specific enzymes of this group to serve distinct roles during the catalytic reaction. An ordered evolutionary adaptation in these enzymes has led to specialization achieved by each enzyme for catalyzing specific reactions. L-Threonine aldolase (L-TA) is one such PLP- dependent enzyme that catalyzes the retro-aldol cleavage of several β-hydroxy amino acids, although its natural substrates are L-threonine and L-allo-threonine with the enzyme having significant preference for L-allo-threonine. It also catalyzes racemization and transamination of D-alanine but not of the β-hydroxy amino acids. Thus, the enzyme exhibits both substrate and reaction specificity. Although, L-TA is frequently employed for stereoselective synthesis of pharmaceutically useful compounds, its reaction mechanism and associated specificity is still not clearly understood. L-TA from Escherichia coli (eTA) is being studied in our laboratory. Our objective is to elucidate the catalytic mechanism of eTA and its mode of substrate and reaction specificity using X-ray crystallography. Another objective is to establish evolutionary relationship of L-TA with other B6-dependent enzymes, such as serine hydroxymethyltransferase (SHMT) and Thermatoga maritima L-TA (TTA) that have the same fold and catalyze similar reactions. Our structural studies show that while the crystal structures of the two L-TAs are similar, they are significantly different from that of SHMT, especially at the active site. In the L-TA structures, a loop with proposed important active site residue, His126 is replaced by tetrahydrofolate (THF) in SHMT. The crystal structures of eTA in its native form and in complex with substrate or product have highlighted the importance of His126 in ensuring substrate specificity during retro-aldol cleavage of various β-hydroxy amino acids and His83 or a conserved water molecule to be active site base. Our study emphasizes the molecular level implications of the catalytic mechanism of eTA.

Introduction: Obese women are predisposed to greater risks of insulin resistance and compensatory hyperinsulinemia. Likewise, African-Americans, appear to be inherently insulin resistant and hyperinsulinemic even after controlling for obesity. Hyperinsulinemia has been attributed to insulin resistance and a compensatory insulin hyper-secretion by the pancreas, as well as decreased insulin clearance, notably in obesity. Pharmacological agents that may worsen insulin resistance/hyperinsulinemia in obese women is of clinical relevance. Previous data from our group suggested that combined oral contraceptives (COCs) may worsen insulin sensitivity particularly in obese women, but limited information on insulin clearance is available in obese women or African-American women. Objective: The objective of the study is to evaluate and compare the effect of a COC containing ethinyl estradiol and norgestimate on insulin clearance among lean and obese pre-menopausal women and among African-American obese vs. non African-American obese women. Method: Plasma insulin clearance was calculated from plasma insulin concentrations, following frequently sampled intravenous glucose tolerance test. Changes in insulin clearance, during six months of COC use were analyzed by repeated measures analysis. Result: Six months of COC use showed no significant change in insulin clearance in all women (p=0.3713). Furthermore, there were no divergent effects on insulin clearance among lean (n=13) and obese (n=14) women (p=0.6703) and among African-American obese (n=7) and non African-American obese (n=7) women (p=0.0957). Changes in insulin clearance, following six months of COC administration was found to be positively correlated with changes in insulin sensitivity (r=0.385, p=0.0099) and negatively correlated with changes in acute insulin response to glucose (r=-0.432, p=0.0034). Discussion: In the present study, COC administration did not show any differential effect on insulin clearance in lean vs. obese women. Future studies evaluating the effects of hormonal agents on insulin-glucose dynamics may focus on mechanisms of hormone-mediated insulin resistance and compensatory hyperinsulinemia rather than insulin clearance.

The discovery of heparin in 1916 resulted in a huge impact on the practice of medicine. Heparin has played a major role in alleviating thrombotic disorders and has also exhibited effects on almost every major system in the human body. Over the past few decades, more and more heparin-protein interactions have come to light. It is implicated to modulate several important processes such as cell growth and differentiation, inflammatory response, viral infection mechanism etc. More interesting is the observation that these interactions are considerably specific with regard to oligosaccharide sequences which have specific spatially oriented sulfate groups modulating the responses. However, due to the complex nature of these interactions and lack of effective computational capabilities, predicting these interactions is challenging.An alternative approach to modulating heparin-protein interactions would be to screen a library of molecules having a diverse distribution of the negative charges and screen them against various proteins of interest to obtain valuable information about the binding/selectivity requirements. This approach would not only yield molecules with potential clinical viability, but may also yield molecules that help decipher native mechanisms regulating proteins, which is called chemical biology in today's terms. Since the difficulties associated with carbohydrate synthesis are well known, well characterized highly sulfated oligosaccharide library screening is considered nearly impossible. Thus, the main aim of this project was to develop an effective method for the synthesis of a library of variably sulfated, non-carbohydrate molecules. The library would contain varying in the number of sulfate groups, offer positional variants of the sulfate groups and provide molecules of varying length so as to afford structural diversity necessary to mimic the heparin sequences. Previous attempts in our laboratory to synthesize such a library encountered two major problems: 1) dimerization of polyphenols due to difficult protection / deprotection strategies and 2) ineffective purification of highly water soluble sulfated molecules. To overcome the problem of protection-deprotection, “click” chemistry has been used in this work for dimerization of polyphenols without any protective groups. To overcome the second problem, a non-aqueous method of purification of highly sulfated molecules was developed, which is the first such report.As a proof of concept, a small library of 14 sulfated monomers and dimers and 8 non-sulfated dimers was generated. The protocol for dimerization of free polyphenolic molecules in has been established to use “click” chemistry for coupling the monomers without the need to protect the free hydroxyl groups. Thus by circumventing the inefficient protection-deprotection protocol, there is a tremendous improvement in yields, ease of purification and characterization and greater productivity allowing the synthesis of more number of molecules in a relatively shorter span of time. By masking the charge of the sulfate using an appropriate counter-ion and owing to the inherent lipophilicity of the aromatic scaffold, these highly charged molecules could be purified using normal phase silica gel chromatography. This method reduced the purification time from previous over 48 hours with the aqueous method to approximately 15 minutes. Further, this purification protocol may be possibly automated so as to truly generate a large library of variably sulfated non-carbohydrate molecules for the first time. Screening this library of 22 sulfated and unsulfated molecules against three enzymes of the coagulation cascade – factors IIa, Xa and XIa – has provided a wealth of information with regard to engineering specificity for recognition of these enzymes. The screening led to the identification of CS3 which inhibited factor XIa with an IC 50 of ~ 5 μM and other enzymes with an IC 50 of > 500 μM as a lead candidate with high selectivity. The success of this strategy bodes well for understanding the heparin-protein interactions at a molecular level. Previous attempts in our laboratory to synthesize such a library encountered two major problems: 1) dimerization of polyphenols due to difficult protection / deprotection strategies and 2) ineffective purification of highly water soluble sulfated molecules. To overcome the problem of protection-deprotection, “click” chemistry has been used in this work for dimerization of polyphenols without any protective groups. To overcome the second problem, a non-aqueous method of purification of highly sulfated molecules was developed, which is the first such report.As a proof of concept, a small library of 14 sulfated monomers and dimers and 8 non-sulfated dimers was generated. The protocol for dimerization of free polyphenolic molecules in has been established to use “click” chemistry for coupling the monomers without the need to protect the free hydroxyl groups. Thus by circumventing the inefficient protection-deprotection protocol, there is a tremendous improvement in yields, ease of purification and characterization and greater productivity allowing the synthesis of more number of molecules in a relatively shorter span of time. By masking the charge of the sulfate using an appropriate counter-ion and owing to the inherent lipophilicity of the aromatic scaffold, these highly charged molecules could be purified using normal phase silica gel chromatography. This method reduced the purification time from previous over 48 hours with the aqueous method to approximately 15 minutes. Further, this purification protocol may be possibly automated so as to truly generate a large library of variably sulfated non-carbohydrate molecules for the first time. Screening this library of 22 sulfated and unsulfated molecules against three enzymes of the coagulation cascade – factors IIa, Xa and XIa – has provided a wealth of information with regard to engineering specificity for recognition of these enzymes. The screening led to the identification of CS3 which inhibited factor XIa with an IC 50 of ~ 5 ?M and other enzymes with an IC 50 of > 500 ?M as a lead candidate with high selectivity. The success of this strategy bodes well for understanding the heparin-protein interactions at a molecular level.