Iniparib nonselectively modifies cysteine-containing proteins in tumor cells and is not a bona fide PARP inhibitor.

PURPOSE: PARP inhibitors are being developed as therapeutic agents for cancer. More than six compounds have entered clinical trials. The majority of these compounds are ß-nicotinamide adenine dinucleotide (NAD(+))-competitive inhibitors. One exception is iniparib, which has been proposed to be a noncompetitive PARP inhibitor. In this study, we compare the biologic activities of two different structural classes of NAD(+)-competitive compounds with iniparib and its C-nitroso metabolite. EXPERIMENTAL DESIGN: Two chemical series of NAD(+)-competitive PARP inhibitors, iniparib and its C-nitroso metabolite, were analyzed in enzymatic and cellular assays. Viability assays were carried out in MDA-MB-436 (BRCA1-deficient) and DLD1(-/-) (BRCA2-deficient) cells together with BRCA-proficient MDA-MB-231 and DLD1(+/+) cells. Capan-1 and B16F10 xenograft models were used to compare iniparib and veliparib in vivo. Mass spectrometry and the (3)H-labeling method were used to monitor the covalent modification of proteins. RESULTS: All NAD(+)-competitive inhibitors show robust activity in a PARP cellular assay, strongly potentiate the activity of temozolomide, and elicit robust cell killing in BRCA-deficient tumor cells in vitro and in vivo. Cell killing was associated with an induction of DNA damage. In contrast, neither iniparib nor its C-nitroso metabolite inhibited PARP enzymatic or cellular activity, potentiated temozolomide, or showed activity in a BRCA-deficient setting. We find that the nitroso metabolite of iniparib forms adducts with many cysteine-containing proteins. Furthermore, both iniparib and its nitroso metabolite form protein adducts nonspecifically in tumor cells. CONCLUSIONS: Iniparib nonselectively modifies cysteine-containing proteins in tumor cells, and the primary mechanism of action for iniparib is likely not via inhibition of PARP activity.

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design.

The DVD-Ig (TM) protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.

Toxicological evaluation of thiol-reactive compounds identified using a la assay to detect reactive molecules by nuclear magnetic resonance.

We have recently reported on the development of a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) to detect reactive false positive hits from high-throughput screening, in which we observed a surprisingly large number of compounds that can oxidize or form covalent adducts with protein thiols groups. In the vast majority of these cases, the covalent interactions are largely nonspecific (e.g., affect many protein targets) and therefore unsuitable for drug development. However, certain thiol-reactive species do appear to inhibit the target of interest in a specific manner. The question then arises as to the potential toxicology risks of developing a drug that can react with protein thiol groups. Here, we report on the evaluation of a large set of ALARM-reactive and -nonreactive compounds against a panel of additional proteins (aldehyde dehydrogenase, superoxide dismutase, and three cytochrome P450 enzymes). It was observed that ALARM-reactive compounds have significantly increased risks of interacting with one or more of these enzymes in vitro. Thus, ALARM NMR seems to be a sensitive tool to rapidly identify compounds with an enhanced risk of producing side effects in humans, including alcohol intolerance, the formation of reactive oxygen species, and drug-drug interactions. In conjunction with other toxicology assays, ALARM NMR should be a valuable tool for prioritizing compounds for lead optimization and animal testing.

Discovery and design of novel HSP90 inhibitors using multiple fragment-based design strategies.

The molecular chaperone HSP90 has been shown to facilitate cancer cell survival by stabilizing key proteins responsible for a malignant phenotype. We report here the results of parallel fragment-based drug design approaches in the design of novel HSP90 inhibitors. Initial aminopyrimidine leads were elaborated using high-throughput organic synthesis to yield nanomolar inhibitors of the enzyme. Second site leads were also identified which bound to HSP90 in two distinct conformations, an 'open' and 'closed' form. Intriguingly, linked fragment approaches targeting both of these conformations were successful in producing novel, micromolar inhibitors. Overall, this study shows that, with only a few fragment hits, multiple lead series can be generated for HSP90 due to the inherent flexibility of the active site. Thus, ample opportunities exist to use these lead series in the development of clinically useful HSP90 inhibitors for the treatment of cancers.

A highly potent and selective farnesyltransferase inhibitor ABT-100 in preclinical studies.

Ras mutation has been detected in approximately 20-30% of all human carcinomas, primarily in pancreatic, colorectal, lung and bladder carcinomas. The indirect inhibition of Ras activity by inhibiting farnesyltransferase (FTase) function is one therapeutic intervention to control tumor growth. Here we report the preclinical anti-tumor activity of our most advanced FTase inhibitor (FTI), ABT-100, and a direct comparison with the current clinical candidates. ABT-100 is a highly selective, potent and orally bioavailable FTI. It broadly inhibits the growth of solid tumors in preclinical animal models. Thus, ABT-100 is an attractive candidate for further clinical evaluation. In addition, our results provide plausible insights to explain the impressive potency and selectivity of ABT-100. Finally, we have demonstrated that ABT-100 significantly suppresses the expression of vascular endothelial growth factor (VEGF) mRNA and secretion of VEGF protein, as well as inhibiting angiogenesis in the animal model.