ATP-Binding Site of Protein Kinases
Dr. Doriano Fabbro,
Novartis, Switzerland
 

Protein kinases play an essential role in many signaling pathways and have the potential to contribute to diseases ranging from cancer and inflammation to diabetes, cardiovascular as well as other disorders. Therefore protein kinases have become one of the most populated class of druggable targets. Identification of kinase specificity and functional validation of kinase as targets has been a challenge, but innovative approaches have been developed to address these issues and over the years a few lessons have been learned:

1. Kinase inhibitors can bind to the kinase by at least four different bind modes:

(i) direct competition with ATP in the ATP binding site;

(ii) engagement of an adjacent allosteric binding site in the ATP pocket usually accessible when the activation loop is in the inactive conformation; and

(iii) binding at sites remote from the ATP site (but still close to the ATP) that impact kinase activity;

(iv) binding outside of the ATP binding pocket (truly allosteric).
 

2. Kinases can escape inhibition by mutating key residues in their catalytic domain and thus become resistant to the kinase inhibitors.

3. Kinase that have gain of function mutations may be more sensitive or resistant to inhibition by kinase inhibitors than the wt form of the kinase.

In this lecture case studies of drug development will be discussed which will provide insights on strategies that are being employed to generate second generation kinase inhibitors including structure-based design, screening approaches, compound selectivity between kinases and the development of the non-ATP competitive kinase inhibitors.

 

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Multiprotein signaling networks create focal points of enzyme activity that disseminate the intracellular action of many hormones and neurotransmitters. Accordingly, the spatio-temporal activation of protein kinases and phosphatases is an important factor in controlling where and when phosphorylation events occur. Anchoring proteins provide a molecular framework that orients these enzymes towards selected substrates. A-kinase anchoring proteins (AKAPs) are signal-organizing molecules that compartmentalize the cAMP dependent protein kinase, phosphodiesterases and a variety of enzymes that are regulated by second messengers. Using a combination of live cell imaging, biochemical, genetic and electrophysiological techniques I will discuss two emerging principles in AKAP signaling: the combinatorial assembly of different enzymes on the same AKAP backbone; and the dynamic reorganization of AKAP complexes.

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The weak binding affinity of DAG for the C1 domains of PKC’s and the complicated structures and restricted supply of natural product ligands prompted us to devise a strategy to enhance the affinity of DAG by means of an optimized DAG-lactone template. DAG-lactones have been designed to selectively direct the flow of information from the DAG signaling pathways through its multiple signal transducers that contain DAG-responsive C1 domains.
 

Because the substrates for PKC phosphorylation and their roles in downstream events are poorly understood, our efforts have been directed to the head of these pathways, at the level of DAG-C1 domain interactions. Since the actual DAG binding site represents a complex of lipid bilayer and C1 domain, for which the C1 domain represents only a half-site, the interactions with the phospholipid head groups are important albeit difficult to predict. We surmised that the specific cellular localization and response of an activated isozyme ought to be determined in part by the different lipid composition of the membranes, and the targeting information intrinsic to the individual isoform. Because structure-activity analyses incorporating the lipid bilayer are still rudimentary, we have decided to explore this aspect indirectly. Thus, we have implemented the syntheses of combinatorial libraries where randomly chosen groups decorating the DAG-lactones generate a series of chemical “zip codes” that create a different PKC-lipid microenvironment capable of directing the activated complex to different sub-cellular sites. Our preliminary results provide a strong proof-of-principle for the concept of chemical “zip codes” which form the basis for new therapeutic strategies selectively targeting these pathways.

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Staurosporine, initially identified as a protein kinase C inhibitor, has turned out to be a highly promiscuous inhibitor. It has inspired a remarkable array of novel compounds, and many highly selective compounds have been found based on this lead structure. We will describe a few aspects of our own foray in this area, in particular with respect to fluoro analogs of LY333531 (ruboxistaurin) and orthoamide analogs of rebeccamycin.
 

Previous results in our laboratories suggested that two conformations dominate in the 14-membered ring macrocyclic bis(indolyl)maleimides. We believe that placing a fluorine atom at the 4-position of one of the indole rings of LY333531 would create an unfavorable lone-pair/lone pair interaction with the carbonyl, and favor a conformation in which the 4-fluorindole ring is perpendicular to the maleimide ring. Indole-fluorinated macrocyclic bisindolylmaleimide have been synthesized to test the hypothesis that this conformation is responsible for the selectivity of LY333531 for the beta isoform of PKC.

Synthetic studies towards staurosporine led us to a new methodology that allowed us to prepare an orthothioamide of bis(indolyl)maleimide. This key intermediate was converted to a family of orthoamide analogs of rebeccamycin. These compounds may allow us to explore the steric, electronic, and conformational space around the key glycosidic bond of the indolocarbazole alkaloids.

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