Design and synthesis of benzoazepin-2-one analogs as allosteric binders targeting the PIF pocket of PDK1.

A novel series of benzoazepin-2-ones were designed and synthesized targeting the PIF pocket of AGC protein kinases, among which a series of thioether-linked benzoazepin-2-ones were discovered to bind to the PIF pocket of 3-phosphoinositide-dependent kinase-1 (PDK1), and to displace the PIF peptide with an EC(50) values in the lower micromolar range. The structure-activity relationships (SARs) of the linker region, tail region, and distal region were explored to further optimize these novel binders which target the PIF pocket of PDK1. When tested in an in vitro PDK1 enzymatic assay using a peptide substrate, the benzodiazepin-2-ones increased the activity of the enzyme in a concentration-dependent fashion, indicating these compounds act as PDK1 allosteric activators. These new compounds may be further developed as therapeutic agents for the treatment of diseases where the PDK1-mediated AGC protein kinases are dysregulated.

An intracellular conformational sensor assay for Abl T315I.

Conformational change is a common molecular mechanism for the regulation of kinase activities. Small molecule modulators of protein conformations, including allosteric kinase inhibitors, are highly wanted as tools for the interrogation of kinase biology and as selective therapeutic agents. However, straightforward cellular assays monitoring kinase conformations in a manner conducive to high-throughput screening (HTS) are not readily available. Here we describe such an HTS-compatible conformational sensor assay for Abl based on a split luciferase construct. The Abl sensor responds to intramolecular structural rearrangements associated with intracellular Abl deactivation and small molecule inhibition. The intact regulatory CAP-SH3-SH2 domain is required for the full functionality of the sensor. Moreover, a T334I Abl mutant (T315I in Abl1a) was found to be particularly well suited for HTS purposes and mechanistic intracellular studies of T334I mutant inhibitors. We expect that the split luciferase-based conformational sensor approach might be more broadly useful to probe the intracellular activation of other kinases and enzymes in general.

Measuring the tyrosine kinase activity: a review of biochemical and cellular assay technologies.

BACKGROUND: The increasing number of kinases as potential drug targets, in combination with the need to screen large compound collections, demands the kinase assays to be homogeneous, non-radioactive, robust, sensitive, easy to miniaturize, and high-throughput. OBJECTIVE: This review will focus on some of the chief biochemical and cellular kinase assay technologies and their applications in tyrosine kinase drug discovery. METHODS: Recent literatures on these tyrosine kinase assay technologies are reviewed, each assay principle, advantages and drawbacks, as well as their potential utilities in tyrosine kinase drug discovery are discussed. RESULTS/CONCLUSION: There is no perfect assay yet; the choice of assay technology relies on the consideration of many factors including the intrinsic properties of the assay, intended application, cost, timeline, synergy with other in-house assay technologies and the expertise, familiarity and comfort level with certain technologies.

Ba/F3 cells and their use in kinase drug discovery.

PURPOSE OF REVIEW: Due to their ability to function as dominant oncogenes, protein kinases have become favored targets in the quest for 'molecularly-targeted' cancer chemotherapeutics. The discovery of a large number of cancer-associated mutations in the kinome, and the progress in developing specific small-molecule kinase inhibitors has increased the need for accurate, reproducible, and efficient kinase activity-dependent cellular assay systems. RECENT FINDINGS: Ba/F3, a murine interleukin-3 dependent pro-B cell line is increasingly popular as a model system for assessing both the potency and downstream signaling of kinase oncogenes, and the ability of small-molecule kinase inhibitors to block kinase activity. Facilitated by their growth properties, Ba/F3 cells have recently been adapted to high-throughput assay formats for compound profiling. Further, several published approaches show promise in predicting resistance to small-molecule kinase inhibitors elicited by point mutations interfering with inhibitor binding. SUMMARY: Ba/F3 cells are an increasingly popular tool in kinase drug discovery. The ability to test the transforming capacity of newly identified kinase mutations, and to profile drug candidates and compound libraries in high-throughput fashion, combined with the use of Ba/F3 cells to predict clinical resistance will greatly facilitate developments in this field.

Design and synthesis of 7H-pyrrolo[2,3-d]pyrimidines as focal adhesion kinase inhibitors. Part 2.

A series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized as focal adhesion kinase (FAK) inhibitors using molecular modeling in conjunction with a co-crystal structure. Chemistry was developed to introduce functionality onto the 9-aryl ring, which resulted in the identification of potent FAK inhibitors. In particular, compound 32 possessed single-digit nanomolar IC(50) and represents one of the most potent FAK inhibitors discovered to date.

An efficient rapid system for profiling the cellular activities of molecular libraries.

Rapid quantitative methods for characterizing small molecules, peptides, proteins, or RNAs in a broad array of cellular assays would allow one to discover new biological activities associated with these molecules and also provide a more comprehensive profile of drug candidates early in the drug development process. Here we describe a robotic system, termed the automated compound profiler, capable of both propagating a large number of cell lines in parallel and assaying large collections of molecules simultaneously against a matrix of cellular assays in a highly reproducible manner. To illustrate its utility, we have characterized a set of 1,400 kinase inhibitors in a panel of 35 activated tyrosine-kinase-dependent cellular assays in dose-response format in a single experiment. Analysis of the resulting multidimensional dataset revealed subclusters of both inhibitors and kinases with closely correlated activities. The approach also identified activities for the p38 inhibitor BIRB796 and the dual src/abl inhibitor BMS-354825 and exposed the expected side activities for Glivec/STI571, including cellular inhibition of c-kit and platelet-derived growth factor receptor. This methodology provides a powerful tool for unraveling the cellular biology and molecular pharmacology of both naturally occurring and synthetic chemical diversity.

Design and synthesis of 7H-pyrrolo[2,3-d]pyrimidines as focal adhesion kinase inhibitors. Part 1.

A series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized to target focal adhesion kinase (FAK). A number of these pyrrolopyrimides exhibited low micromolar inhibitory activities against focal adhesion kinase, and their preliminary SAR was established via systematic chemical modifications. The 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines represent a new class of kinase inhibitors.

Identification of coumarin derivatives as a novel class of allosteric MEK1 inhibitors.

A homogenous TR-FRET-based in vitro coupling assay for the MAP3Ks-MEK1-ERK2 kinase cascade was established and was used to screen for inhibitors of the ERK/MAPK pathway. A series of coumarin derivatives were identified from the screen. These compounds potently inhibit the activation of the unactivated human MEK1 by upstream MAP3Ks (including BRAF and COT), but do not inhibit the activity of the activated MEK1. In addition, the potency of these compounds in inhibiting MEK1 activation is not affected by varying the ATP concentration, suggesting that these inhibitors are not competitive with ATP. As expected, the coumarin compounds potently inhibit LPS-induced TNFalpha production and ERK phosphorylation in THP-1 cells, with the most potent compound having an IC(50) of 90nM. Molecular modeling studies suggest that these coumarins bind to an allosteric site in the inactive conformation of MEK1. This site has been shown to be utilized by the biarylamine series of MEK inhibitors such as PD318088. Very interestingly, the identified coumarin derivatives are almost identical to a series of inhibitors recently reported that block LPS-induced TNFalpha production. Our findings have therefore raised the possibility that other naturally occurring or synthetic coumarins with anti-cancer and anti-inflammatory activities might exert their biological function through the inhibition of MEK1.

Crystal structures of human HSP90alpha-complexed with dihydroxyphenylpyrazoles.

A series of dihydroxyphenylpyrazole compounds were identified as a unique class of reversible Hsp90 inhibitors. The crystal structures for two of the identified compounds complexed with the N-terminal ATP binding domain of human Hsp90alpha were determined. The dihydroxyphenyl ring of the compounds fits deeply into the adenine binding pocket with the C2 hydroxyl group forming a direct hydrogen bond with the side chain of Asp93. The pyrazole ring forms hydrogen bonds to the backbone carbonyl of Gly97, the hydroxyl group of Thr184 and to a water molecule, which is present in all of the published HSP90 structures. One of the identified compounds (G3130) demonstrated cellular activities (in Her-2 degradation and activation of Hsp70 promoter) consistent with the inhibition of cellular Hsp90 functions.

A time-resolved fluorescence resonance energy transfer-based HTS assay and a surface plasmon resonance-based binding assay for heat shock protein 90 inhibitors.

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone required for the stability and function of a number of client proteins, many of which are involved in cancer development. The natural products geldanamycin (GM) and radicicol (RD) are known inhibitors of Hsp90, and their derivatives are being developed for the treatment of various cancers. To identify novel Hsp90 inhibitors, a highly robust time-resolved fluorescence resonance energy transfer (TR-FRET)-based HTS assay that measures the binding of biotinylated geldanamycin (biotin-GM) to the His-tagged human Hsp90 N-terminal ATP-binding domain (Hsp90N) was developed. This assay was optimized in 1536-well plates and was used as the primary assay to screen 10(6) compounds. Identified "hits" were then confirmed in a scintillation proximity assay (SPA) and a DEAE membrane-based assay for [(3)H]AAG binding to Hsp90. In addition, a surface plasmon resonance (SPR) assay that measures the direct interaction of Hsp90 with its inhibitors was developed and used to further characterize the identified inhibitors. Several potent and reversible inhibitors of human Hsp90 with K(d) values measured in the high nanomolar range were identified.