Structure-Based Design of Selective LONP1 Inhibitors for Probing In Vitro Biology.

LONP1 is an AAA+ protease that maintains mitochondrial homeostasis by removing damaged or misfolded proteins. Elevated activity and expression of LONP1 promotes cancer cell proliferation and resistance to apoptosis-inducing reagents. Despite the importance of LONP1 in human biology and disease, very few LONP1 inhibitors have been described in the literature. Herein, we report the development of selective boronic acid-based LONP1 inhibitors using structure-based drug design as well as the first structures of human LONP1 bound to various inhibitors. Our efforts led to several nanomolar LONP1 inhibitors with little to no activity against the 20S proteasome that serve as tool compounds to investigate LONP1 biology.

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.

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.

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.

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.

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.

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.