This project has focused on exploring the application of Baylis-Hillman (BH) {a.k.a. Morita-Baylis-Hillman (MBH)} scaffolds in the construction of various compounds with medicinal potential. A series of 2-nitrobenzaldehydes has been treated under BH conditions, with two different activated alkenes, viz., (MVK) and methyl acrylate, using (DABCO) or (3-HQ) as catalyst. While most of the BH reactions were carried out at room temperature, some reactions were conducted using microwave irradiation. The resulting BH adducts have been subjected to dehydration, conjugate addition and allylic substitution to obtain appropriate intermediates, which have been used in turn, to synthesize possible lead compounds, viz., cinnamate esters as HIV-1 integrase inhibitors, 3-(aminomethyl)quinolines and quinolones as anti-malarials and cinnamate ester-AZT conjugates as dual-action HIV-1 integrase-reverse transcriptase (IN-RT) inhibitors. Conjugate addition reactions of methyl acrylate-derived BH β-hydroxy esters with the amines, piperidine, propargylamine and 2-amino-5-(diethylamino)pentane, has afforded a range of products as diastereomeric mixtures in moderate to excellent yields. Catalytic hydrogenation of the aminomethy β-hydroxy esters derivatives, using a palladium-oncarbon (Pd-C) catalyst, has afforded the corresponding, novel 3-aminomethyl-2- quinolone derivatives in moderate yields. Effective allylic substitution reactions of the MVK-derived BH β-hydroxy ketones (via a conjugate addition-elimination pathway) using in situ-generated HCl has afforded the corresponding α-chloromethyl derivatives, which have been reacted with various amines, including piperidine, piperazine, propargylamine and 2-amino-5-(diethylamino)pentane, to yield α-aminomethyl derivatives. Catalytic hydrogenation of selected α-aminomethyl derivatives, using a Pd-C catalyst, has afforded the corresponding, novel 3- (aminomethyl)-2-methylquinoline derivatives in low to moderate yields. A bioassay, conducted on a 6-hydroxy-2-methyl-3-[(piperidin-1-yl)methyl]quinoline isolated early in the study indicated anti-malarial activity and prompted further efforts in the synthesis of analogous compounds. Reaction of the methyl acrylate-derived BH adducts with POCl3 has provided access to α-(chloromethyl)cinnamate ester derivatives, which have been aminated to afford α- (aminomethyl)cinnamate ester derivatives as potential HIV-1 integrase inhibitors. The α- (propargylaminomethyl)cinnamates were used, in turn, as substrates for the “click chemistry” reaction with 3'-azido-3'-deoxythymidine (AZT– an azide and an established reverse transcriptase HIV-1 inhibitor) to afford cinnamate ester-AZT conjugates as potential dual-action HIV-1 integrase-reverse transcriptase (IN-RT) inhibitors. Computer modelling and docking studies of a cinnamate ester-AZT conjugate into the HIV-1 integrase and reverse transcriptase active-sites revealed potential hydrogen-bonding interactions with amino acid residues within the receptor cavities. The isolated products have been appropriately characterized using IR, 1- and 2-D NMR and HRMS techniques, while elucidation of the stereochemistry of the double bond in the BH-derived halomethyl derivatives has been assigned on the basis of NOE, computer modelling and X-ray crystallographic data.

Water soluble platinum complexes are a recent area of emphasis of cisplatin chemistry. The water soluble complexes could have a reduced toxicity compared with cisplatin. Oxaliplatin, which has an oxalate leaving group, has previously been shown to have less nephro-toxicity and higher water solubility than cisplatin. [Pt(en)(oxalate)] (en = ethylenediamine) has been prepared from Pt(en)Cl2 and silver oxalate. This complex has been reacted with methionine and N-acetylmethionine at different molar ratios. At high Pt: methionine ratios, chelates with the sulfur and nitrogen atoms of the methionine are dominant; at lower Pt: methionine ratios, a bis-methionine product is formed. The en ligand is displaced by methionine but not N-acetylmethionine.

Parasitic diseases remain a health burden affecting more than 500 million people worldwide with malaria having the highest mortality rate. The parasites can be transferred to the human bodies either through the mouth by ingestion of contaminated food and water or through the skin by bug bites or direct contact to environments harbouring them. Epidemiological control seems to be impossible since there is failure to control the insect vectors as well as practice of hygiene. Therefore, this has led to the development of a number of vaccines, chemotherapy and disease control programs. However, parasites have increasingly developed resistance to traditionally used anti-parasitic drugs and due to that fact there is need for alternative medication for parasitic diseases. Heat shock protein 90 (Hsp90) facilitates the folding of proteins in all living cells and their role is more important to parasites because of their environmental changes, from vector to host. Hsp90s play a major role; therefore this justifies the need for a deeper analysis of the parasitic Hsp90s. Recent studies have revealed that, the Plasmodium sp. Hsp90 has an extended linker region which increases the protein’s affinity for ATP and its inhibitors. Therefore we hypothesize that there are also significant features in other parasitic Hsp90s which would lead to Hsp90 being defined as potential drug targets. In the present study an attempt was made to gain more insight into the differences in primary structure of human and parasitic Hsp90s. The sequences were retrieved from the NCBI database and analysis was done in three groups basing on the localization of the Hsp90. The physicochemical properties were calculated and in every group, the protozoan Hsp90s showed significant differences when compared to the human orthologs. Multiple sequence alignments (MSA) showed that endoplasmic reticulum Hsp90s have an extended region in the middle domain indicating their ability to bind to a unique subset of client proteins. Sequence identities between the human and parasites showed that the protozoan Hsp90s are less related to the human Hsp90s as compared to the other parasites. Likewise, motif analysis showed the trypanosomatids and apicomplexan groups have their own unique set of motifs and they were grouped together in the phylogenetic analysis. Phylogenetic analysis also showed that, the protozoan Hsp90s forms their own clades in each group while the helminths did not form in endoplasmic reticulum group. In this study, we concluded that, Hsp90 can be a potential drug target for the protozoan species and more specifically those from the apicomplexan and trypanosomatids groups.

Current chemotherapeutics pose many di sadvantages due to their lack of specificity and low therapeutic index. To overcome these challenges, research has focused its attention on the development of nano-based delivery systems that can penetrate the leaky vasculature of tumor endothelium, use site-directed ligands that can bind with high affinity and specific ity to tumor cells, physically entrap poorly soluble drugs, and deliver these cytotoxic agents directly to the tumor site. One approach to nanosystem drug delivery is with the use of peptide amphiphiles (PAs) that are conjugated with the Arginine-Glycine-Aspartic Acid (RGD) motif to actively target a αVβ3 integrin receptors on cancer cells or tumor endothelium. The current work is focused on mechanistic studies to evaluate the uptake of novel RGD amphiphi les with varying alkyl chain lengths (palmitic acid : Cl 6 and stearic acid: C 18) and hydrophilic linkers, 8-amino- 3,6-dioxaoctonoic acid (ADA) or glucose, as micellar delivery systems of hydrophobic anticancer agents. PAs were confirmed for their self-assembling properties and further evaluated for their RGD-mediated binding specificity to purified αVβ3 integrin through a competitive binding fluorescence polarization assay (with novel RGD micelles displacing an integrin-bound fluorescent RGD probe by as much as 63.03%). Ultimately, these nanocarriers were assessed for their ability to deliver phys ically entrapped fluorescein isoth iocyanate (FITC) to A2058 cells overexpressing αVβ3 integrin receptors. Results from confocal microscopy indicate that uptake of RGD micelles was driven by an energy-dependent mechanism, as statistically significant levels of FITC internalization was seen at 37°C versus 4°C (p-value<0.05 for all treatment groups); moreover, intracellular fluorescence was notably higher (as much as 4-fold) when delivered through novel RGD conjugates as opposed to its free form. Regardless of chain length and the number of hydrophilic linkers, all RGD PAs showed promising results as micellar carriers that can effectively deliver their payload to the target tumor site via receptor mediated endocytosis.

Chemotherapy persists as one of the mainstays of breast cancer treatment, particularly for triple-negative breast cancer which currently has no targeted treatment methods. While chemotherapy is beneficial for killing the malignant tumor cells, it leads to the release of damage-associated molecular patterns into the tumor microenvironment. Damage-associated molecular patterns are a contributing factor to cancer-related inflammation which can potentiate metastatic spread through several mechanisms such as the development of tumor microenvironments at metastastic sites. These damage-associated molecular patterns include nucleic acids, nucleic acid-associated lipids and vesicles, cytokines, and proteins such as high mobility group protein B1. Polyamidoamine (PAMAM) is a biodegradable, water-soluble dendrimer polymer with the ability to possess different charges and sizes depending on its terminal branches and degree of branching (i.e. generation number), respectively. Amine-terminated PAMAM-NH2 is positively charged and can bind to circulating DNA and RNA. Since most DAMP molecules are negatively charged, I hypothesized that a polycation such as PAMAM-NH2 would be an efficient nanomaterial to remove pathogenic NA DAMPs generated by chemotherapy. Building on this dendrimer, we synthesized modified cationic PAMAM-generation 3 derivatives with an aim to balance toxicity with NA-binding affinity and capacity to encapsulate chemodrugs. Our results found that these soluble and nanoparticle PAMAM materials can bind to both cell-free DNA and RNA released as a result of treating triple-negative breast cancer cells with chemotherapy drugs such as doxorubicin and paclitaxel. These PAMAM-G3 materials are termed as nucleic acid binding polymers and nucleic-acid binding polymeric nanoparticles.My thesis dissertation explores the anti-metastatic effects of nucleic-acid binding polymeric nanoparticles delivering the chemotherapy drug paclitaxel using in-vitro and in-vivo models. Two murine metastatic breast cancer models served as the basis for assessing the effects of conventional paclitaxel delivery compared to paclitaxel delivery from within PAMAM nucleic-acid binding polymeric nanoparticles with respect to primary tumor growth, extent of lung metastasis, and the systemic inflammatory response reflected in murine serum. Compared to treatment with unencapsulated paclitaxel, delivery of paclitaxel within the PAMAM nucleic-acid binding polymeric nanoparticles resulted in significantly decreased serum cell-free DNA levels, decreased inflammatory cytokines, and a lower degree of lung metastasis in the mice. The decrease in the degree of lung metastasis in mice receiving paclitaxel within the PAMAM nanoparticles was confirmed by assessing the photon flux signal of 4T1-luciferase breast cancer cells invading the murine lungs in both in-vivo and ex-vivo imaging and by using a machine learning method to quantify the degree of metastasis in H&E- stained sections of the lungs. The ability to mitigate the phenomenon of chemotherapy-induced cancer metastasis while effectively delivering the chemotherapy to the tumor microenvironment could help improve the outcomes of patients being treated with chemotherapy. This work developed a therapeutic cationic PAMAM nanocarrier-based strategy to inhibit paclitaxel-induced metastasis by scavenging cell-free nucleic acids and mitigating cell-free nucleic acid-induced inflammation.

Indiana University-Purdue University Indianapolis (IUPUI) / The antiretroviral drug efavirenz remains a cornerstone for treatment-naïve HIV patients. Subsequent to the demonstration that efavirenz is a substrate of cytochrome P450 (CYP) 2B6, a number of clinical studies found that the CYP2B6*6 allele is significantly associated with higher efavirenz exposure and/or adverse reactions. However, the mechanism of reduced efavirenz metabolism by this genetic variant is not fully understood and whether this variant exhibits differential susceptibility to metabolic inhibition is also unknown. Ths use of efavirenz is further complicated by the drug interactions associated with it. Therefore, I hypothezised that 1) the CYP2B6*6 allele reduces efavirenz metabolism by altering catalytic properties of CYP2B6; 2) efavirenz alters the pharmacokinetics of co-administered drugs by inhibiting drug metabolizing enzymes. A series of studies was carried out in hepatic microsomal preparations to determine the functional consequences of the CYP2B6*6 allele and to assess inhibition potency of efavirenz on 8 CYPs. The major findings for these studies include: 1) the CYP2B6*6 allele reduces efavirenz metabolism by decreasing substrate binding and catalytic efficiency; 2) functional consequences of the CYP2B6*6 allele appear to be substrate- and cytochrome b5-dependent; 3) the CYP2B6*6 allele confers increased susceptibility to metabolic inhibition; and 4) efavirenz inhibits the activities of CYP2B6, 2C8, 2C9 and 2C19 at therapeutically relevant concentrations. In addition, I explored the hypothesis that the incorporation of in vitro mechanism by which the CYP2B6*6 allele reduced efavirenz metabolism predicts the genetic effect of this allele on efavirenz clearance after a single oral dose by modeling approach. A pharmacogenetics-based in vitro-in vivo extrapolation (IVIVE) model was developed to predict human efavirenz clearance. Taken together, results from this dissertation provide new mechanistic information on how the CYP2B6*6 allale alters substrate metabolism and drug interactions; demonstrate new mechanisms of efavirenz-mediated inhibition interactions; and demonstrate the utility of a pharmacogenetics-based predictive model that can serve as a basis for future studies with efavirenz and other CYP2B6 substrates. Overall these data provide improved understanding of genetic and non-genetic determinant of efavirenz disposition and drug interactions associated with it.

Indiana University-Purdue University Indianapolis (IUPUI) / All platinum (Pt)-based chemotherapeutics exert their efficacy primarily via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of Pt-DNA lesions by nucleotide excision repair and homologous recombination (HR) can substantially reduce the effectiveness of the Pt therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions requires RPA binding to single-stranded DNA (ssDNA). We synthesized structural analogs of our previously reported RPA inhibitor TDRL-505, determined the structure activity relationships and evaluated their efficacy in tissue culture models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro and cellular activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.