Discovery and characterization of non-ATP site inhibitors of the mitogen activated protein (MAP) kinases.

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.

Biochemical and biophysical characterization of unique switch pocket inhibitors of p38α.

Herein we describe the identification and characterization of a class of molecules that are believed to extend into a region of p38 known as the 'switch pocket'. Although these molecules lack a canonical hinge binding motif, they show K(i) values as low as 100 nM against p38. We show that molecules that interact with this region of the protein demonstrate different binding kinetics than a canonical ATP mimetic, as well as a wide range of kinome profiles. Thus, the switch pocket presents new opportunities for kinome selectivity which could result in unique biochemical responses and offer new opportunities in the field of kinase drug discovery.

In search of negative allosteric modulators of biological targets.

The value of using negative allosteric modulators of protein function in therapeutic treatment of human diseases is becoming more apparent. Many current screening paradigms, however, are not consciously designed to discover negative allosteric modulators, and those that are serendipitously discovered can be easily overlooked during the hit-picking process. The conditions necessary for the discovery of negative allosteric modulators in a high-throughput screen are quite reasonable and simple to implement, generally requiring a consideration of the ligand concentration in a screen. Other considerations in the screening for negative allosteric modulators can be derived from an analysis of simple kinetic schemes that describe the interactions of ligands and modulators with different protein targets.

Screening for positive allosteric modulators of biological targets.

The benefit of using positive allosteric modulators of protein function in the therapy of human diseases is becoming more apparent. The advantage of positive allosteric modulators is that they can possess specificity and selectivity profiles as well as concentration-independent limits on activity that can significantly reduce off-target effects in vivo. However, many current screening paradigms are not designed to discover positive allosteric modulators, and modulators that are discovered serendipitously can be overlooked during the hit-picking process. The conditions needed to discover positive allosteric modulators in a HTS are reasonable and simple to implement, generally requiring consideration of the ligand concentration in a screen. Other considerations in the screening for positive allosteric modulators can be derived from the analysis of simple kinetic schemes that describe the interactions of ligands and modulators with different protein targets.

Functional analysis of large conductance Ca2(+)-activated K(+) channels: ion flux studies by atomic absorption spectrometry.

Although techniques such as (86)Rb(+) flux provide a sensitive measure of K(+) channel activity, the relatively short half-life and high-energy emission, together with the quantities of radioactive material generated, hinder the usefulness of flux-based formats in high throughput screening efforts. This study elaborates on the utilization of flame atomic absorption spectrometry (AAS) techniques for a nonradioactive rubidium efflux assay for large conductance Ca(2+)-activated K(+) channels (BK(Ca)) channels. Utilizing human embryonic kidney (HEK293) cells expressing the BK(Ca) alpha subunit, a 96-well cell-based nonradioactive rubidium efflux screen for channel openers and inhibitors was established. Known BK(Ca) channel openers, including NS1608, NS1619, and NS-8, activated rubidium efflux with EC(50) values ranging from 1 to 4 microM in both radioactive and nonradioactive efflux formats. Compounds such as iberiotoxin, paxilline, and charybdotoxin inhibited rubidium efflux responses evoked by the BK(Ca) channel opener NS1608 in both radioactive and nonradioactive efflux formats. The IC(50) values of the inhibitors in AAS format were comparable to those derived from (86)Rb(+) efflux assays. The present studies show that the pharmacological profiles of BK(Ca) channels assessed by AAS compare well with those obtained using the (86)Rb(+) efflux assay, and support the utility of nonradioactive efflux format for higher throughput screening campaigns for novel K(+) channel modulators.

Putting thought to paper: a microARCS protease screen.

In micro-arrayed compound screening (microARCS), an agarose gel is used as a reaction vessel that maintains humidity and compound location as well as being a handling system for reagent addition. Two or more agarose gels may be used to bring test compounds, targets, and reagents together, relying on the pore size of the gel matrix to regulate diffusion of reactants. It is in the microenvironment of the agarose matrix that all the components of an enzymatic reaction interact and result in inhibitable catalytic activity. In an effort to increase the throughput of microARCS-based screens, reduce the effort involved in manipulating agarose gels, and reduce costs, blotter paper was used rather than a second agarose gel to introduce a substrate to a gel containing a target enzyme. In this assay, the matrix of the blotter paper did not prevent the substrate from diffusing into the enzyme gel. The compound density of the microARCS format, the ease of manipulating sheets of paper for reagent addition, and a scheduled protocol for running multiple gels allowed for a throughput capacity of more than 200,000 tests per hour. A protease assay was developed and run in the microARCS format at a rate of 200,000 tests per hour using blotter paper to introduce the substrate. Picks in the primary screen were retested in the microARCS format at a density of 384 compounds per sheet. IC(50) values were confirmed in a 96-well plate format. The screen identified several small molecule inhibitors of the enzyme. The details of the screening format and the analysis of the hits from the screen are presented.

Functional characterization of adenosine receptors and coupling to ATP-sensitive K+ channels in Guinea pig urinary bladder smooth muscle.

Although multiple adenosine receptors have been identified, the subtype and underlying mechanisms involved in the relaxation response to adenosine in the urinary bladder remain unclear. The present study investigates changes in the membrane potential, as assessed by fluorescence-based techniques, of bladder smooth muscle cells by adenosine receptor agonists acting via ATP-sensitive potassium (K(ATP)) channels. Membrane hyperpolarization evoked by adenosine and various adenosine receptor subtype-selective agonists was attenuated or reversed by the K(ATP) channel blocker glyburide. Comparison of adenosine receptor agonist potencies eliciting membrane potential effects showed a rank order of potency 5'-N-ethyl-carboxamido adenosine (NECA; -log EC50 = 7.97) approximately 2-p-(2-carboxethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680; 7.65) > 2-chloro adenosine (5.90) approximately 2-chloro-N6-cyclopentyladenosine (CCPA; 5.51) approximately N6-cyclopentyladenosine approximately N6-(R)-phenylisopropyladenosine > 2-chloro- N6-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2Cl-IBMECA; 4.78). Membrane potential responses were mimicked by forskolin, a known activator of adenylate cyclase, and papaverine, a phosphodiesterase inhibitor. The A(2A)-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino] ethyl)phenol (ZM-241385), and the adenylate cyclase inhibitor N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride (MDL-12330A) inhibited the observed change in membrane potential evoked by adenosine and adenosine-receptor agonists. The rank order potency for relaxation of K+-stimulated guinea pig bladder strips, NECA (-log EC50 = 6.41) approximately CGS-21680 (6.38) > 2-chloro adenosine (5.90) >> CCPA approximately 2Cl-IBMECA (>4.0) was comparable to that obtained from membrane potential measurements. Collectively, these studies demonstrate that adenosine-evoked membrane hyperpolarization and relaxation of bladder smooth muscle is mediated by A(2A) receptor-mediated activation of K(ATP) channels via adenylate cyclase and elevation of cAMP.