Prema Iyer

Mutagenicity of N3-methyladenine: A multi-translesion polymerase affair

We recently demonstrated that Polzeta and Rev1 contribute to alleviate the lethal effects of Me-lex, which selectively generates 3-methyladenine, by error prone lesion bypass. In order to determine the role of Poleta in the biological fate of Me-lex induced lesions, the RAD30 (Poleta) gene was deleted in the yIG397 parental strain and in its rev3 (Polzeta) derivative, and the strains transformed with plasmid DNA damaged in vitro by Me-lex. While deletion of RAD30 increased the toxicity of Me-lex, the impact on mutagenicity varied depending on the concentration of Me-lex induced DNA damage and the overall TLS capacity of the cells. For the first time the Me-lex induced mutation spectrum in rad30 strain was determined and compared with the spectrum previously determined in WT strain. Overall, the two mutation spectra were not significantly different. The effect on mutation frequency and the features of the Me-lex induced mutation spectra were suggestive of error prone (significant decrease of mutation frequency and significant decrease of AT>TA at a mutation hotspot in rad30 vs RAD30) but also error free (significant increase of AT>GC in rad30 vs RAD30) Poleta dependent bypass of lesions. In summary, our previous results with Polzeta and Rev1 mutants, the present results with Poleta, and the known physical and functional interactions among TLS proteins, lead us to propose that the bypass of Me-lex induced lesions is a multi-DNA polymerases process that is mostly effective when all three yeast TLS polymerases are present.

Synthesis and Characterization of DNA Minor Groove Binding Alkylating Agents

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.

Design and evaluation of novel glutaminase inhibitors.

A novel set of GAC (kidney glutaminase isoform C) inhibitors able to inhibit the enzymatic activity of GAC and the growth of the triple negative MDA-MB-231 breast cancer cells with low nanomolar potency is described. Compounds in this series have a reduced number of rotatable bonds, improved ClogPs, microsomal stability and ligand efficiency when compared to the leading GAC inhibitors BPTES and CB-839. Property improvements were achieved by the replacement of the flexible n-diethylthio or the n-butyl moiety present in the leading inhibitors by heteroatom substituted heterocycloalkanes.

High frequency of genomic deletions induced by Me-lex, a sequence selective N3-adenine methylating agent, at the Hprt locus in Chinese hamster ovary cells

We have investigated the mutagenicity induced at the Hprt locus in Chinese hamster ovary (CHO) cells treated with increasing concentrations of Me-lex, a minor groove selective methylating agent that efficiently generates more than 90-95% of 3-MeA DNA adducts. Me-lex treatment was cytotoxic but weakly mutagenic, resulting in up to 7-fold induction above background in the Hprt mutation frequency. The molecular nature of 43 Hprt mutations induced by Me-lex was determined by sequence analysis of the Hprt cDNA and genomic analysis of the gene locus. Base pair substitutions represented about 25% of Me-lex induced mutations. The mutation spectrum revealed a high percentage of genomic deletions (51%) comprising single/multiple exon(s) and even the loss of the complete locus. When the distribution of mutations among different classes was considered, the difference between the spontaneous and Me-lex induced CHO spectra was statistically significant (p<0.012), indicating that the sites where mutations occurred were Me-lex specific. Based upon these results we hypothesize that a large proportion of mutations may result from the processing of 3-MeA, the main adduct induced by Me-lex, within A/T rich sequences in non-coding regions of the Hprt gene. The processing of these lesions by DNA polymerases could result in recombination and genomic deletions, thus representing a severe threat for genome integrity.

Characterization of the interactions of potent allosteric inhibitors with glutaminase C, a key enzyme in cancer cell glutamine metabolism

Altered glycolytic flux in cancer cells (the “Warburg effect”) causes their proliferation to rely upon elevated glutamine metabolism (“glutamine addiction”). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl (BPTES), and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared with BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We also show that UPGL00004 potently inhibits the growth of triple-negative breast cancer cells, as well as tumor growth when combined with the anti-vascular endothelial growth factor antibody bevacizumab. Finally, we compare the X-ray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism. These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism.

Connecting Neuronal Cell Protective Pathways and Drug Combinations in a Huntington’s Disease Model through the Application of Quantitative Systems Pharmacology

Quantitative Systems Pharmacology (QSP) is a drug discovery approach that integrates computational and experimental methods in an iterative way to gain a comprehensive, unbiased understanding of disease processes to inform effective therapeutic strategies. We report the implementation of QSP to Huntington’s Disease, with the application of a chemogenomics platform to identify strategies to protect neuronal cells from mutant huntingtin induced death. Using the STHdhQ111 cell model, we investigated the protective effects of small molecule probes having diverse canonical modes-of-action to infer pathways of neuronal cell protection connected to drug mechanism. Several mechanistically diverse protective probes were identified, most of which showed less than 50% efficacy. Specific combinations of these probes were synergistic in enhancing efficacy. Computational analysis of these probes revealed a convergence of pathways indicating activation of PKA. Analysis of phospho-PKA levels showed lower cytoplasmic levels in STHdhQ111 cells compared to wild type STHdhQ7 cells, and these levels were increased by several of the protective compounds. Pharmacological inhibition of PKA activity reduced protection supporting the hypothesis that protection may be working, in part, through activation of the PKA network. The systems-level studies described here can be broadly applied to any discovery strategy involving small molecule modulation of disease phenotype.

XRCC1 deficiency influences the cytotoxicity and the genomic instability induced by Me-lex, a specific inducer of N3-methyladenine.

Me-lex is a sequence-specific alkylating agent synthesized to preferentially (>90%) generate N3-methyladenine (3-mA) in the minor groove of double-strand DNA, in A-T rich regions. In this paper we investigated the effect of XRCC1 deficiency in the processing of 3-mA adducts generated by Me-lex, through the molecular analysis of the Hprt mutations and the evaluation of cytogenetic end points such as sister chromatid exchanges (SCEs), micronuclei (MN) and nucleus fragmentation. EM-C11 cells, deficient in XRCC1 activity, showed a 2.5-fold higher sensitivity to the toxicity of Me-lex compared to the DNA repair proficient parental CHO-9 cells, but were not hyper mutable. The spontaneous mutation spectrum at the Hprt locus generated in EM-C11 cells revealed a high percentage of genomic deletions. After Me-lex treatment, the percentage of genomic deletions did not increase, but a class of mutations which appeared to target regulatory regions of the gene significantly increased (p=0.0277), suggesting that non-coding Hprt genomic sequences represent a strong target for the rare mutations induced by Me-lex. The number of SCEs per chromosome increased 3-fold above background in 50mucapital EM, Cyrillic Me-lex treated CHO-9 cells, while at higher Me-lex concentrations a sharp increase in the percentage of MN and fragmented nuclei was observed. In EM-C11 cells the background level of SCEs (0.939+/-0.182) was approximately 10-fold higher than in CHO-9 (0.129+/-0.027) and higher levels of multinucleated cells and MN were also found. In EM-C11, even low doses of Me-lex (25microM) led to a significant increase in genomic damage. These results indicate that XRCC1 deficiency can lead to genomic instability even in the absence of an exogenous genotoxic insult and low levels of Me-lex-induced lesions, i.e., 3-mA and/or a BER intermediate, can exacerbate this instability.