Allosteric Small-Molecule Serine/Threonine Kinase Inhibitors.

Deregulation of protein kinase activity has been linked to many diseases ranging from cancer to AIDS and neurodegenerative diseases. Not surprisingly, drugging the human kinome - the complete set of kinases encoded by the human genome - has been one of the major drug discovery pipelines. Majority of the approved clinical kinase inhibitors target the ATP binding site of kinases. However, the remarkable sequence and structural similarity of ATP binding pockets of kinases make selective inhibition of kinases a daunting task. To circumvent these issues, allosteric inhibitors that target sites other than the orthosteric ATP binding pocket have been developed. The structural diversity of the allosteric sites allows these inhibitors to have higher selectivity, lower toxicity and improved physiochemical properties and overcome drug resistance associated with the use of conventional kinase inhibitors. In this chapter, we will focus on the allosteric inhibitors of selected serine/threonine kinases, outline the benefits of using these inhibitors and discuss the challenges and future opportunities.

Sterically controlled C-H/C-H homocoupling of arenes via C-H borylation.

A mild one-pot protocol for the synthesis of symmetrical biaryls by sequential Ir-catalyzed C-H borylation and Cu-catalyzed homocoupling of arenes is described. The regiochemistry of the biaryl formed is sterically controlled as dictated by the C-H borylation step. The methodology is also successfully extended to heteroarenes. Some of the products obtained by this approach are impossible to obtain via the Ullmann or the Suzuki coupling protocols. Finally, we have shown a one-pot sequence describing C-H borylation/Cu-catalyzed homocoupling/Pd-catalyzed Suzuki coupling to obtain π-extended arene frameworks.

Allosteric Targeting of Aurora A Kinase Using Small Molecules: A Step Forward Towards Next Generation Medicines?

BACKGROUND: Aurora A (AurA) kinase is a key mitotic protein implicated in cancer. Several small molecule inhibitors targeting the ATP binding site of this enzyme are in various stages of clinical development. However, these inhibitors can result in selectivity and drug resistance problems. Allosteric inhibition of kinases using small molecules is an alternative strategy to target these enzymes selectively and these could serve as the seeds for next generation medicines. This review discusses the developments in the non-ATP site binding small molecule inhibitors of AurA and their prospect as future therapeutics. DISCUSSION: Allosteric targeting of AurA kinase using small molecules is relatively a new strategy, and only a handful of research work has been reported. Two patents and three papers pertaining to allosteric targeting of AurA kinase using small molecules were covered in this review. Topics discussed include, identification of small molecule inhibitors targeting AurA- Targeting Protein for Xenopus kinesin-like protein 2 (TPX2) interaction, anacardic acid - a natural product ligand that selectively modulates AurA activity in the presence of Aurora B kinase, and identification of felodipine as an uncompetitive inhibitor of AurA using Surface Enhanced Raman Spectroscopy (SERS) technique. CONCLUSION: Allosteric targeting of therapeutically relevant enzymes using small molecules is a burgeoning research area. New techniques such as fragment-based ligand discovery, SERS methods, etc., are expanding to identify the allosteric site binding ligands. Research in this area is expected to deliver fruitful outcome in terms of novel therapeutics against AurA kinase as well as other therapeutically relevant enzymes.

Activity-based fingerprinting and inhibitor discovery of cysteine proteases in a microarray.

A panel of 20 peptide vinyl sulfone probes has been synthesized and used to generate activity-based fingerprinting profiles of cysteine proteases in both gel- and microarray-based formats; the inhibitor fingerprints of representative small molecule inhibitors targeted against 4 cysteine proteases were also obtained, in high-throughput, using the same protein microarray platform.

Activity based fingerprinting of proteases using FRET peptides.

We have successfully developed a protease assay using fluorescence resonance energy transfer based peptide libraries, which allows not only general detection of enzymatic activities, but more importantly substrate fingerprinting of proteases from different classes. The method allows the generation of substrate fingerprints of a protease from both the nonprime and prime sites. Therefore, it is well suited for profiling of major metalloproteases such as thermolysin and MMPs. We envisage that this method will provide a useful tool in the emerging field of Catalomics for high-throughput studies of proteases.

Advanced analytical tools in proteomics.

Proteomics deals with the study of proteins, their structures, localizations, posttranslational modifications, functions and interactions with other proteins. The mapping of protein structure-function holds the key to a better understanding of cellular functions under both normal and disease states, which is critical for modern drug discovery. However, the study of human proteome presents scientists with a task much more daunting than the human genome project. In fact, the estimated >100,000 different proteins expressed from 30,000 to 40,000 human genes make it extremely challenging, if not impossible with existing protein analysis techniques, to map the entire cellular functions at the translational level. Consequently, there have been rapid advances in the techniques and methods capable of large-scale proteomic studies. Among them, the recently developed high-throughput screening methods have enabled scientists to analyze proteins quickly and efficiently at an organism-wide scale. Herein, we overview some of these emerging tools for high-throughput protein analysis. In particular, we focus on recent advances in the bioassay development, which has provided sensitive and selective tools for high-throughput identification and characterizations of enzymes. Finally, the recently developed bioimaging techniques to visualize and quantify proteins in living cells are also discussed.

A proteomic strategy for the identification of caspase-associating proteins.

We report the efficient in vivo labelling of caspases expressed inside apoptotic HeLa cells using fluoromethyl ketone (fmk)-containing probes; preliminary results indicated that these probes may be used to identify caspase-associating proteins.

Developing photoactive affinity probes for proteomic profiling: hydroxamate-based probes for metalloproteases.

The denaturing aspect of current activity-based protein profiling strategies limits the classes of chemical probes to those which irreversibly and covalently modify their targeting enzymes. Herein, we present a complimentary, affinity-based labeling approach to profile enzymes which do not possess covalently bound substrate intermediates. Using a variety of enzymes belonging to the class of metalloproteases, the feasibility of the approach was successfully demonstrated in several proof-of-concept experiments. The design template of affinity-based probes targeting metalloproteases consists of a peptidyl hydroxamate zinc-binding group (ZBG), a fluorescent reporter tag, and a photolabile diazirine group. Photolysis of the photolabile unit in the probe effectively generates a covalent, irreversible linkage between the probe and the target enzyme, rendering the enzyme distinguishable from unlabeled proteins upon separation on a SDS-PAGE gel. A variety of labeling studies were carried out to confirm that the affinity-based approach selectively labeled metalloproteases in the presence of a large excess of other proteins and that the success of the labeling reaction depends intimately upon the catalytic activity of the enzyme. Addition of competitive inhibitors proportionally diminished the extent of enzyme labeling, making the approach useful for potential in situ screening of metalloprotease inhibitors. Using different probes with varying P(1) amino acids, we were able to generate unique "fingerprint" profiles of enzymes which may be used to determine their substrate specificities. Finally, by testing against a panel of yeast metalloproteases, we demonstrated that the affinity-based approach may be used for the large-scale profiling of metalloproteases in future proteomic experiments.

Recent advances in peptide-based microarray technologies.

Peptide array is a rapidly growing tool that provides both large-scale and high-throughput capabilities for protein detection and activity studies. Materials presented in this review will examine the recent advances in the field of peptide microarray with special emphasis on the generation and applications of high-density arrays of peptides on glass slides.

Strategies for immobilization of biomolecules in a microarray.

Recent advances in the generation of peptide and protein microarrays are reviewed, with special focuses on different strategies available for site-specific immobilization of proteins and peptides.