Browsing by Subject "Medicinal chemistry"
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Item Design and synthesis of tubulysin analogs that stabilize and mimic a key acetate important for potent antiproliferative activity against multi-drug resistant cancers(2012-10) Peterson, Michael ThomasTubulysins are antimitotic natural products with potent anticancer activity against multidrug-resistant (MDR) cancer cells, acting by inhibition of tubulin polymerization. The marked difference in antiproliferative activity between tubulysins V and U exemplifies the importance of an acetate positioned alpha to the thiazole ring. However, this acetate has been shown to be labile under both acidic and basic conditions, so the effectiveness of this modification may be hindered due to this instability. Hence, the work presented here focuses on the synthesis of analogs that mimic and stabilize the acetate at this position.Heteroatom exchange at the α-thiazole position of tubuvaline was hypothesized to increase molecular stability while maintaining observed activity by bioisosteric replacement of the tubuvaline oxygen with a nitrogen. The nitrogen-containing analogs of tubulysin V and U, N tubulysin V and U, were the most important targets to test the singular modification of heteroatom exchange on bioactivity and to survey molecular stability. The synthetically derived N tubuvaline amino acid residue was generated following a rigorously controlled Mitsunobu reaction, but difficult final stage deprotections to N tubulysin V suggests a lowered stability compared to tubulysin V. N-acylation of a penultimate tetrapeptide intermediate led to generation of N tubulysin U and other acylated N tubulysin analogs to establish a more robust SAR at the α-thiazole position. N Tubulysin U was found to be more stable than tubulysin U under strongly basic conditions, and upcoming biochemical evaluation will determine the effect that these modifications have on antiproliferative activity.Investigations into the SAR at the tubuvaline α-thiazole position also included oxygen-based analogs, where two methods for acylation of tubulysin V were exploited to generate O-acylated analogs with various alkyl groups. Biochemical evaluation of antiproliferative activity, along with the use of two electrophilic analogs to act as affinity labels, will survey important interactions within the tubulysin-tubulin binding site.Item Fragment based inhibitor design of Mycobacterium tuberculosis BioA(2015-01) Dai, Ran7,8-Diaminopelargonic acid synthase (BioA) of Mycobacterium tuberculosis (Mtb) is a recently validated target for therapeutic intervention in the treatment of tuberculosis (TB). We herein report our fragment based inhibitor design of Mtb BioA. Using differential scanning fluorimetry (DSF) fragment screening, the Maybridge Ro3 library of 1000 molecules was screened. Twenty-one compounds giving rise to Tm shifts exceeding ±2°C were then investigated in crystallographic experiments. Six fragments have been co-crystallized with BioA to characterize binding. Each compound has a unique binding mode, and subtle variations in ligand binding site geometry are induced upon binding of different fragment molecules. Binding affinities of the fragments were characterized via isothermal titration calorimetry (ITC). A fragment extension strategy was used to rationally optimize these fragment hits. A commerce based SAR was used and identified 50 compounds containing the core of one of the fragments. These compounds were further screened virtually and experimently by DSF. Four optimized BioA ligands from fragment optimization were validated by X-ray crystallography, including a potent aryl hydrazine inhibitor of BioA that reversibly modifies the pyridoxal-5′-phosphate (PLP) cofactor. Binding affinities of these ligands have been characterized by ITC or kinetic assay. The six fragment complex structures were also used for optimization of HTS lead compounds. Six HTS lead compounds were co-crystallized with BioA at high resolution. Design of optimized compounds was by overlapping the fragments and HTS lead binding conformations in the BioA active site. Molecules predicted to have better potency were proposed. Two N-aryl piperazine inhibitors of BioA from HTS optimization were characterized using X-ray crystallography and ITC. One inhibitor that combines features of one HTS lead and one fragment was confirmed with improved binding affinity by ITC.Item I. DNA-protein cross-linking by CIS-1,1,2,2-diamminedichloroplatinum (II) (cisplatin) II. formation of 8-OXO-dG and oxazolone lesions with p53 II. derived DNA sequences following photooxidation in the presence of riboflavin(2011-05) Ming, Xun1,1,2,2- Cis-diamminedichloroplatinum (II) (cisplatin) is a common anticancer drug used in treatment of solid tumors. The biological activity of cisplatin is generally attributed to its ability to form DNA-DNA cross-links by sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also forms a variety of DNA-protein cross-links (DPCs) whose structures are not well characterized. While the biological outcomes of cisplatin-induced DPC lesions are not understood, they are hypothesized to interrupt important cellular processes such as DNA replication and transcription, potentially leading to toxicity. In the present work, a human DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate cisplatin-induced DNA-protein cross-linking. The normal physiological function of AGT is to repair alkylation damage by transferring O6-alkylguanine groups from DNA to an active site cysteine residue of the protein (Cys145), restoring normal guanine. Incubation of recombinant AGT protein with 32P-labeled oligonucleotides duplexes in the presence of cisplatin resulted in concentration-dependent formation of DNA-protein conjugates as revealed by denaturing gel electrophoresis. Capillary HPLC-electrospray ionization mass spectrometry analysis (ESI-MS) of AGT protein treated with dG-Pt-Cl monoadduct as a model of monoplatinated DNA confirmed the ability of the protein to form multiple dG-AGT cross-links. Upon heating, dG-Pt-AGT complexes undergo platination migration from protein to the N7 position of guanine to form dG-Pt-dG cross-links. This can be explained by greater thermodynamic stability of the Pt-N bond as compared to the Pt-S bond. HPLC-ESI+-MS/MS sequencing of tryptic peptides derived from dG-Pt-AGT complexes revealed that cisplatin-mediated cross-linking involves six different sites within this protein: Glu110, Lys125, Cys145, His146, Arg147, and Cys150. Among these, Cys145, His146, Arg147, and Cys150 are located in the protein active site and directly or indirectly participate in alkyl transfer. Finally, HPLC-ESI+ - MS/MS analysis of total proteolytic digests detected 1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2’-deoxyguanosine-7-yl)-platinum (II) (dG-Pt-Lys) conjugates produced via platination of lysine residues within AGT. To identify protein targets of cisplatin-induced cross-linking in nuclear protein extracts from human cervical carcinoma (HeLa) cells, an affinity capture methodology was combined with mass spectrometry-based proteomics and immunological detection. A total of 131 nuclear proteins were identified to form covalent DPCs in the presence of cisplatin. An estimated DNA-protein cross-linking efficiency following treatment with 50 μM cisplatin was 2-16%, depending on protein identity. HPLC-ESI+-MS/MS analysis of total proteolytic digests of cross-linked proteins revealed the presence of dG-Pt-Lys conjugates. We further extended this work to characterize DNA-protein cross-linking by cisplatin in human fibrosarcoma (HT1080) cells. Following drug treatment, DPCs were isolated by a modified phenol/chloroform DNA extraction incorporating proteasome inhibitors. Proteins were released from DNA by heating and identified by mass spectrometry-based proteomics and immunological detection. Over 250 nuclear proteins were found to be captured on chromosomal DNA following treatment with cisplatin. HPLC-ESI+-MS/MS analysis of total proteolytic digests revealed the formation of dG-Pt-Lys conjugates between the N7 guanine of DNA and the ε-amino group of lysine. Although cisplatin-induced DPCs spontaneously release proteins to form DNA-DNA cross-links upon heating, they appear to be stable enough under physiological conditions to inhibit DNA replication and transcription, contributing to the biological effects of cisplatin. These results indicate that clinically relevant concentrations of cisplatin induce covalent cross-links between chromosomal DNA and a large range of nuclear proteins. If not repaired, the resulting bulky DPC lesions are likely to contribute to both on-target and off-target toxicity of cisplatin. Reactive oxygen species produced as part of normal cellular metabolism and immune response can damage cellular DNA, giving rise to promutagenic nucleobase lesions. Since the biological impact of a given oxidative adduct is influenced by its position within gene sequence, previous studies have focused on determining the distribution of oxidative lesions along DNA sequences. However, since these studies have relied on gel electrophoresis to locate the sites of oxidative damage, they could not analyze the distribution of structurally defined nucleobase lesions and suffered from the high background of direct strand breaks induced by sugar oxidation. We now report the use of stable isotope labeling of DNA-mass spectrometry (ILD-MS) approach to map the formation of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG) and 2,2-diamino-4-[2-deoxy-β-D-erythro-pentofuranosyl)amino]-2,5-dihydrooxazol-5-one (oxazolone) lesions along DNA sequences derived from the p53 tumor suppressor gene. In each duplex, one of the guanine bases was labeled with [1,7,NH2-15N3-2-13C]-guanine which served as an isotope “tag” to enable specific quantification of guanine lesions originating from that position. Following photooxidation in the presence of riboflavin, DNA was enzymatically digested to 2’-deoxynucleosides, and the formation of 8-oxo-dG and oxazolone at each site of interest was quantified from isotope ratios obtained from capillary HPLC-ESI+-MS/MS. We found that in double stranded DNA, both oxidative lesions were generated non-randomly, and their distribution was strongly influenced by the local DNA sequence. In particular, the 5’ Gs in guanine repeats and guanines within MeCG dinucleotides were preferentially targeted for photooxidation in the presence of riboflavin. This can be explained by the low ionization potential of 5’-guanine at 5’-GG sites and/or the preferential intercalation of riboflavin at MeCG sites. Furthermore, the most frequently adducted position, G5 in exon 5, G4 and G7 in exon7, and G6 in exon 8, coincide withthe known p53 lung cancer mutational “hotspots” at p53 codons 158 (CGC), 245 (GGC), 248 (CGG), and 273 (CGT), respectively, suggesting that oxidative DNA damage may contribute to mutagenesis in the p53 gene.Item Mass spectrometry-based analysis of urinary metabolites of 1,3-Butadiene (BD) in humans and influence of BD-DNA adducts on DNA replication(2013-10) Kotapati, SrikanthCigarette smoking is a known risk factor for the development of lung cancer: approximately 1 out of 5 heavy smokers will develop the disease. However, there are significant differences in risk of lung cancer among smokers from different ethnic/racial groups. African American and Native Hawaiian smokers are at a higher risk of lung cancer than European American, Japanese American or Latin American smokers. Cigarette smoke has more than 70 known carcinogens. Following metabolic activation to electrophilic species, these carcinogens can form covalent DNA adducts, which are capable of inducing heritable mutations ultimately resulting in lung cancer. It has been hypothesized that the observed ethnic/racial differences in lung cancer risk in smokers are due to different frequencies of specific polymorphisms in drug metabolizing genes, leading to a different degree of carcinogen bioactivation to DNA-reactive intermediates. 1,3-Butadiene (BD) is among the most abundant and potent carcinogens present in cigarette smoke. BD is metabolically activated primarily by CYP2E1 to form 3,4-epoxy-1-butene (EB), hydroxymethyl vinylketone (HMVK), 3,4-epoxy-1,2-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB). EB, HMVK, EBD, and DEB have been shown to modify DNA bases to form promutagenic adducts. Alternatively, EB, EBD, and DEB can undergo detoxification via epoxide hydrolysis (the main pathway in humans) or glutathione conjugation and further metabolic conversion into urinary mercapturic acids, 1-hydroxy 2-(N-acetylcysteinyl)-3-butene (MHBMA), 1,2-dihydroxy-4-(N-acetyl cysteinyl)-butane (DHBMA), 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), and 1,4-bis-(N-acetylcysteinyl)butane-2,3-diol (bis-BDMA), respectively. The research presented in this thesis focuses on revealing any ethnic/racial differences in metabolism of BD in smokers and examining the ability of BD-DNA adducts to cause mutations. In the first part of the thesis, we have identified two novel metabolites of BD which have not been previously detected in vivo: 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA), and 1,4-bis-(N-acetylcysteinyl)butane-2,3-diol (bis-BDMA). To enable their detection in smokers, sensitive and specific HPLC-ESI--MS/MS methods were developed for both metabolites in human urine. We observed significant amounts of THBMA in samples from smokers, non-smokers and occupationally exposed workers. In contrast, bis-BDMA amounts in urine of smokers and occupationally exposed workers were below the method's limit of detection, although it was found in urine of F344 rats exposed to 62.5 or 200 ppm BD. Additionally we found significant interspecies differences in BD metabolism between laboratory rats and humans. DHBMA accounted for only 47% of BD urinary mercapturic acids in rats while the corresponding percentage in humans is 93%. We further developed a high throughput HPLC-ESI--MS/MS method for the quantification of MHBMA and DHBMA in humans and applied this method to quantify urinary BD-mercapturic acids metabolites in workers from a BD and styrene butadiene rubber (SBR) manufacturing facility and smokers belonging to different ethnic groups in two separate multi-ethnic cohort studies. Workers occupationally exposed to BD excreted significantly more BD-mercapturic acids than administrative workers at the same plant. In a small multi-ethnic study of smokers belonging to European American, Native Hawaiian and Japanese American (N = 200 per group), mean urinary MHBMA and MHBMA/DHBMA+MHBMA metabolic ratio were highest in European American and lowest in Japanese American smokers. Similar results were obtained in the larger study (N = 450 per group) composed of European American and African American smokers. Urine of European American smokers contained higher concentrations of MHBMA than that of African Americans. Genome-wide association study (GWAS) analysis conducted for the larger multi-ethnic group has revealed significant associations between single-nucleotide polymorphisms (SNPs) in chromosome 22 (22564172bp - 22735492 bp, nearby genes GSTT1, GSTT2, DDT and MIF) and urinary BD-mercapturic acid levels in smokers, providing the first evidence for genetic and ethnic/racial differences in metabolism of BD.The second part of my thesis work has focused on evaluating the mutagenic ability of three recently discovered BD-dA lesions: N6-(2-hydroxy-3-buten-1-yl)-adenine (N6-HB-dA), N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB-dA), and 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-&gamma-HMHP-dA). In vitro translesion synthesis experiments were performed on synthetic oligonucleotides containing each of the three lesions at a site-specific position by gel electrophoresis and HPLC-MS/MS. We found that human translesion synthesis (TLS) polymerases hPols &eta, &kappa, &iota and human polymerase &beta were able to bypass (S)-N6-HB-dA in an error-free manner because of the conserved Watson-Crick base pairing with dT. However, replication past both (R,R)-N6,N6-DHB-dA and (R,S)-1,N6-&gamma-HMHP-dA lesions by TLS polymerases hPols &eta and &kappa was highly error-prone, resulting in A&rarrT, A&rarrC mutations and frameshift deletions. This is the first study that identifies (R,R)-N6,N6-DHB-dA and (R,S)-1,N6-&gamma-HMHP-dA as BD-DNA adducts potentially responsible for the induction of A*rarrT mutations by BD.Item Self-assembled nanostructures for the cellular delivery of small molecules, oligonucleotides and proteins for the treatment of cancer(2014-02) Gangar, AmitWe have developed chemically self-assembled nanostructures (CSANs) as a platform to achieve targeted drug delivery. CSANs are composed of dihydrofolate reductase (DHFR) and its inhibitor methotrexate. We have demonstrated that DHFR nanorings spontaneously self assemble in a controlled manner, when two fused DHFR molecules (DHFR2) are mixed with two chemically attached methotrexate (bis-MTX) molecules. Targeting CSANs selectively to the cancer cells was achieved by fusing one of the DHFR molecules with a single chain variable fragment (scFv) of an antiCD3 antibody. Delivery of antiCD3 scFv functionalized CSANs to CD+ leukemia T-cells have been demonstrated. The next step was to exploit the targeting ability of CSANs towards delivery of payload molecules such as drugs, nucleic acids and imaging agents. The research presented in the thesis focuses on evaluating the CSANs for its ability to achieve targeted drug delivery. In the first part of the thesis, we have engineered DHFR-DHFR dimer interface to drive the equilibrium towards formation of preferential heterodimers of DHFR. We have performed a series of point mutations at the dimer interface and screened mutants for the formation homodimeric and heterodimeric species. We have demonstrated that 65 % heterodimers of DHFR could be obtained by this method compared to 50 % obtained when protein mixed in the stoichiometric proportion. The second part of the thesis focuses on CSANs mediated targeted delivery of oligonucleotides. CSANs were functionalized with antisense oligonucleotides (ASO) targeting eukaryotic translation initiation factor 4E (eIF4E) via bis-MTX. Targeting was achieved using cyclic RGD peptide that binds to the alfa-v beta-3 integrin receptors. We have demonstrated that ASO bearing RGD CSANs bind to the alfa-v beta-3 integrin positive breast cancer cells, undergo receptor-mediated endocytosis and knock down the expression of eIF4E protein. In the third part, we have assembled bispecific CSANs that engage the immune cells to attack the cancerous cells. Bispecifc CSANs are composed of DHFR2antiCD3 scFv that binds to the CD3 receptor on immune T-cells and DHFR2 linked to a small peptide that target the epitopes on cancer cell surface. In the current study, we have targeted epithelial growth factor receptor (EGFR) that is over-expressed on several solid tumors. We observed about 70 % killing of EGFR positive glioblastoma cells over 24 hrs when incubated with T-cells that were functionalized with bispecific CSANs. In the last part of the thesis, we have evaluated targeted CSANs for their in-vivo biodistribution in mice bearing human tumors. Our preliminary studies demonstrate higher uptake of the CSANs by organs such as the liver, pancreas and kidneys primarily due to the macrophage uptake. To overcome this problem we have site-specifically PEGylated the CSANs using non-natural amino acid mutagenesis. In the in-vitro studies, we have demonstrated that PEGyaltion of CSANs does not affect their cancer cell binding ability but significantly reduced their macrophage uptake. In addition, we have also performed the biodistribution of PEGylated CSANs in mice tumor models.Item Synthesis and evaluation of parthenolide analogues: chemical probes and therapeutic agents(2013-03) Wang, DanCancer stem cells (CSCs), also known as tumor-propagating cells or tumor-initiating cells, are subpopulations of undifferentiated, highly tumorigenic cells found within bulk tumors. The rapid advances of cancer research and development of relative technologies have provided more and more evidence for the existence of CSCs, as well as the important roles they play in drug resistance and disease relapse of cancer. However, because of their quiescent nature and the similarities to normal stem cells, eradicating CSCs presents a challenging task. Chapter one provides an overview of cell surface markers of CSCs. Those markers are potential diagnostic macromolecules and targets for drug delivery.Parthenolide (PTL) is a sesquiterpene lactone natural product isolated from Mexican Indian medicinal herb Tanacetum parthenum (feverfew plant), a known medical herb utilized for centuries. PTL has been extensively studied as an anticancer agent, showing significant efficacy towards a wide spectrum of human cancer cells. In 2005, the identification of PTL as the first stand-alone and selective cytotoxic agent against the acute myeloid leukemia CSCs further heightened its therapeutic potential. However, the mechanism of action of PTL's CSC inhibitory activity is still an area of debate. Our efforts to elucidate the molecular targets of PTL is described in chapter two. The design and synthesis of two PTL affinity probes with diverse biological activity as well as their utilization in comparative and competitive protein pull-down experiments to enrich the cellular protein targets of PTL is presented. Although exhibiting promising anticancer and anti-CSC activities, the modest biological potency and poor water solubility prevent further development of PTL. Chapter three describes our efforts to synthesize PTL analogues, as well as our strategy to prepare water-soluble PTL prodrugs.