Discovery of a potent and selective Bcl-2 inhibitor using SAR by NMR.

The Bcl-2 family of proteins plays a major role in the regulation of apoptosis, or programmed cell death. Overexpression of the anti-apoptotic members of this family (Bcl-2, Bcl-x(L), and Mcl-1) can render cancer cells resistant to chemotherapeutic agents and therefore these proteins are important targets for the development of new anti-cancer agents. Here we describe the discovery of a potent, highly selective, Bcl-2 inhibitor using SAR by NMR and structure-based drug design which could serve as a starting point for the development of a Bcl-2 selective anti-cancer agent. Such an agent would potentially overcome the Bcl-x(L) mediated thrombocytopenia observed with ABT-263.

Aminopyridine-based c-Jun N-terminal kinase inhibitors with cellular activity and minimal cross-kinase activity.

The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP) kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety of different diseases with an inflammatory component including asthma, stroke, Alzheimer's disease, and type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range, activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1,000-fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38alpha, and p38delta and showed little inhibitory activity against a panel of 74 kinases.

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.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.

Protein fragmentation via liquid chromatography-quadrupole time-of-flight mass spectrometry: the use of limited sequence information in structural characterization.

Fragmentation and "top-down" sequencing of intact proteins by mass spectrometry (MS) is most commonly performed by infusion of protein solutions into Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. However, the high cost of this instrumentation, coupled with the need to infuse "clean" solutions (lacking standard biological buffers), limits broad application of this technique. The current study describes an alternative approach to top-down sequencing using in-source fragmentation on quadrupole time-of-flight (Q-Tof) instrumentation coupled with reversed-phase liquid chromatography (LC). Application of this technique to purified recombinant samples yielded protein fragments during routine LC-MS analysis. The presence of multiple N- and C-terminal fragments allowed localization of structural modifications without proteolytic digestion. The method was extended to complex samples by using LC conditions that provided high-resolution protein separation. Utility of the method was illustrated by real-time monitoring of protein modifications occurring in reconstituted apoptosomes. These experiments illustrate that intact protein mass and limited sequence information can be obtained simultaneously on an LC timescale. This approach will allow a wide variety of laboratories to routinely apply top-down sequencing to problems in structural characterization, protein purification, and biomarker identification.