High affinity protein kinase inhibitors

Details High affinity protein kinase inhibitors High affinity protein kinase inhibitors

2000-01-11

2002-05-07

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S184000, C544S262000, C514S261100

Reexamination Certificate

active

06383790

ABSTRACT:

FIELD OF THE INVENTION
This invention provides general methods for discovering mutant inhibitors for any class of enzymes as well as the specific inhibitors so identified. More specifically, this invention provides general methods for discovering specific inhibitors for multi-substrate enzymes. Examples of such multi-substrate enzymes include, but are not limited to, kinases and transferases. The mutant inhibitors identified by the methods of this invention can be used to highly selectively disrupt cell functions such as oncogenic transformation. In one particular example, this invention provides a Src protein kinase inhibitor, pharmaceutical compositions thereof and methods of disrupting transformation in a cell that expresses the target v-Src comprising contacting the cell with the protein kinase inhibitor.
BACKGROUND OF THE INVENTION
All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. U.S. patent application Ser. Nos. 08/797,522 and 60/046,727, and PCT/US98/02522 are related to the present invention and each of these applications is specifically and individually incorporated by reference in its entirety.
The current explosion in the number of newly discovered genes underscores the need for small molecule ligands which can be used to elucidate and control gene function. Convergent engineering of protein/small molecule interfaces has emerged in recent years as a powerful method for generating novel ligand/receptor pairs with high specificity. By introducing chemical diversity into the target protein as well as the small molecule, unique binding interactions can be designed and exploited more efficiently than through traditional medicinal chemistry. Such approaches have been used to chemically explore a number of biological systems. FK506-binding protein has been engineered to preferentially bind non-natural FK506 analogues by Schreiber and co-workers, as well as Clackson and co-workers. This system has been used extensively to selectively dimerize receptors and control gene expression in a cellular context. Nuclear hormone receptors have also been shown to be amendable to chemical genetic design. Corey and co-workers demonstrated that mutations at two amino acid residues in the retinoid X receptor are sufficient to create two new classes of receptors with novel ligand specificities. In a more medicinally applicable system, Smith and co-workers engineered the protease, carboxypeptidase A1, to hydrolyze a prodrug of methotrexate which is resistant to hydrolysis by wild type proteases.
Protein kinase catalyzed phosphorylation of the hydroxyl moiety of serine, threonine or tyrosine is the central post-translational control element in eukaryotic signal transduction. The phosphorylation state of a given protein can govern its enzyme activity, protein-protein binding interactions, and cellular distribution. Phosphorylation and dephosphorylation is thus a “chemical switch” which allows the cell to transmit signals from the plasma membrane to the nucleus to ultimately control gene expression in a highly regulated manner. Highly selective, cell permeable inhibitors of individual kinases would allow for the systematic investigation of the cellular function of a kinase in real time, and thus, would provide invaluable tools for the deconvolution of phosphorylation dependent processes in signal transduction cascades.
The Src family is composed of ten highly homologous cytosolic kinases which are critical components in an array of cell signaling pathways ranging from lymphocyte activation to cell growth and proliferation. Constitutive activation of these enzymes can lead to oncogenic cell transformation, making them putative drug targets for cancer therapies. Because of their importance in the regulation of these fundamental cellular processes, many studies have focused on developing inhibitors for the Src family kinase. However, the potent inhibitors that have been discovered lack the high selectivity that would be required for probing the cellular inhibition of an individual target kinase. Conventional inhibitor screens have produced few if any molecules which can discriminate between the active sites of the various Src family kinases.
Unfortunately, the very features which make kinases so useful in signal transduction, and which has made them evolve to become central to almost every cellular function, also makes them extremely difficult, if not impossible, to study and understand. Their overlapping protein specificities, their structural and catalytic similarities, their large number, and their great speed make the specific identification of their in vivo protein substrates extremely difficult, if not impossible, using current genetic and biochemical techniques. This is today the main obstacle to deciphering the signaling cascades involved in protein kinase-mediated signal transduction (4,6-8).
Efforts to dissect the involvement of specific protein kinases in signal transduction cascades have been frustrated by their apparent lack of protein substrate specificity in vitro and in vivo (4,8). The catalytic domains of protein kinases possess little or no inherent protein substrate specificity, as demonstrated by domain swapping experiments (18-23). The catalytic domain from one protein kinase can be substituted into a different protein kinase with little change in the protein substrate specificity of the latter (22).
The poor in vitro specificity of kinases also makes it difficult, if not impossible, to extrapolate what the in vivo function of given kinases might be. An isolated protein kinase of interest will often phosphorylate many test substrates with equal efficiency (29). This apparently poor substrate specificity is also found in vivo; for example, many genetic approaches, such as gene knock out experiments, give no interpretable phenotype due to compensation by other cellular protein kinases (30,31).
Another complication is that many protein kinases have been proposed to phosphorylate downstream and upstream proteins which are themselves protein kinases; although this appears to make complex positive feedback loops possible, it also makes dissecting the cascade even more difficult (1). One important avenue for deciphering the role and understanding the function of enzymes, both in vitro and in vivo, is the use of specific enzyme inhibitors. If one or more compound can be found that will uniquely inhibit the protein kinase target, the inhibitor can be used to modulate the enzyme's activity, and the effects of that decrease can be observed. Whole genome techniques have provided many targets but their function is unknown. Many methods have been developed to determine if a given new kinase could be a good target. These methods, all have in common the lack of a small molecule to inhibit the enzyme which can lead to confusion.
For example, the most commonly used state of the art technique is to knock out the kinase and see a new phenotype. Typically, deletion of one kinase in the mouse genome (most common model organism) causes no informative change. This is for two reasons: 1) either the gene kinase) is essential during embryogenesis, thereby causing lethality before birth, or 2) the gene is absent (knocked out) and its function can be replaced by a closely related kinase which is still present. The important difference between the art recognized approach and the invention herein is that herein small organic molecules are employed to inhibit the function of the kinase of interest, since it is still present in the organisms but inactive thus it can cause significant changes to the organisms and most importantly the changes are exactly like that which would occur if an inhibitor of wild-type enzyme was made.
In addition, such inhibitors are among the most important pharmaceutical compounds known. For example, aspirin (acetylsalicylic acid) is such an inhibitor. It inhibits an enzyme that catalyzes the first s

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