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.

7,8-dihydroxyflavone (7,8-DHF), which is a member of the flavonoid family, is a selective tyrosine kinase receptor B (TrkB) agonist that has neurotrophic effects in various neurological diseases such as ischemic stroke and Parkinson’s disease [3]. In this study, we assessed the angiogeneic effect of 7,8-DHF in endothelial cells derived from resistance vessel of the brain. Angiogenesis by 7,8-DHF is an important factor that helps prevent and treat various ischemic diseases. In this study, we found that rat RV cells used in the experiment possess the TrkB receptor. Our data also demonstrates that 7,8-DHF is able to stimulate cell proliferation in RV cells, suggesting that 7,8-DHF is capable of inducing angiogenesis. The 7,8-DHF activates the TrkB receptor which then leads to cell proliferation. In our study we also showed the effects of 7,8-DHF in the presence of the TrkB inhibitor cyclotraxin-B. Addition of cyclotraxin-B blocked the TrkB receptor and counteracted the effects of 7,8-DHF. Cell proliferation occurs in RV cells with the addition of 7,8-DHF, but this proliferation is inhibited by cyclotraxin-B.

Flavonoids are ubiquitous to all terrestrial plants and many are known to have anti-tumor activities. In this research project we studied the differential cytotoxic effects of two flavone isomers on human breast cancer cells (BT-474, SKBR-3, and MCF-7) and normal breast cells as a control (MCF-10A). The basis of the relationships between chemical structures, type and position of substituent groups and the effects these compounds exert specifically on cancer cells are not completely elucidated. These flavones are derived from plants native of Colombia, South America. These plants of the family Asteraceae, genera Gnaphalium and Achyrocline are reputed to have anti-cancer properties. The flavones are non-toxic to normal human cells. Our studies indicate that 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone, flavone B) is highly cytotoxic to poorly differentiated carcinomas of the breast such as SK-BR3, with minimal activity against more differentiated carcinomas of the same organs such BT-474. Immunoblot analysis suggests that the anti-tumor effects of flavone B in SKBR-3 may be mediated through the down regulation of the ERK pathway. ERK activation promotes cell proliferation, differentiation and survival. Additionally, flavone B down regulates PS6 in SKBR-3. PS6 controls protein translation by phosphorylating the S6 protein of the 40S ribosomal subunit. On the other hand, neither flavone A or B has a significant inhibitory effect on breast cancer cell line BT-474. The flavones were tested in the human fibrocystic mammary tissue MFC-10A, defined as a normal breast cell line, to demonstrate their lack of toxicity against normal cells.

Abstract available in the Journal of Oncology Pharmacy Practice.

Oral antineoplastics (OAs) have become an emerging and rapidly growing field in cancer treatment. As with any chemotherapeutic agent, clinicians must be aware of potential drug interactions. Drug information databases are a common resource utilized to check for interactions between OAs and patient’s home medications. A major concern with OAs is that they are usually taken at home as well as picked up at a pharmacy by the patient themselves. The objective of this study was to determine the reliability of these databases for identifying potential interactions with OAs in a real-world setting. Hospital records were used to identify patients with common malignancies (leukemia, sarcoma, colon, lung, thyroid, prostate, kidney and liver cancers) treated with OAs from 2013 to 2014. To be included patients must have started an OA during the study period and have adequate records to evaluate OA use as well as home medications. The patient’s regimen is then entered into Drugs.com and Lexicomp™ interaction databases. In addition to documenting the number of interactions flagged by both databases, the severity of the interaction and disagreements between databases were analyzed. A major interaction was defined as either a “D” or “X” by Lexicomp™ and “major” by Drugs.com. As of this preliminary analysis, 407 of 876 subjects have been screened. Of the 407 screened, 9 patients (one patient with 3 different OAs) have been enrolled. Lexicomp™ flagged 34 interactions, of which 10 were major interactions. Drugs.com flagged 34 interactions, of which 6 were major interactions. Between the 2 databases there was only 60% agreement in flagging major interactions. These discrepancies are of concern in that clinicians hope resources they utilize are congruent with one another and allow them to practice in the safest manner in terms to avoid clinically significant drug interactions OA.