Todd W. Mayhood

Selective small-molecule inhibition of an RNA structural element.

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

Crystal Structures of MEK1 Binary and Ternary Complexes with Nucleotides and Inhibitors.

MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. We have determined that the kinase domain spans residues 35-382 by proteolytic cleavage. The complete kinase domain has been crystallized and its X-ray crystal structure as a complex with magnesium and ATP-gammaS determined at 2.1 A. Unlike crystals of a truncated kinase domain previously published, the crystals of the intact domain can be grown either as a binary complex with a nucleotide or as a ternary complex with a nucleotide and one of a multitude of allosteric inhibitors. Further, the crystals allow for the determination of costructures with ATP competitive inhibitors. We describe the structures of nonphosphorylated MEK1 (npMEK1) binary complexes with ADP and K252a, an ATP-competitive inhibitor (see Table 1), at 1.9 and 2.7 A resolution, respectively. Ternary complexes have also been solved between npMEK1, a nucleotide, and an allosteric non-ATP competitive inhibitor: ATP-gammaS with compound 1 and ADP with either U0126 or the MEK1 clinical candidate PD325089 at 1.8, 2.0, and 2.5 A, respectively. Compound 1 is structurally similar to PD325901. These structures illustrate fundamental differences among various mechanisms of inhibition at the molecular level. Residues 44-51 have previously been shown to play a negative regulatory role in MEK1 activity. The crystal structure of the integral kinase domain provides a structural rationale for the role of these residues. They form helix A and repress enzymatic activity by stabilizing an inactive conformation in which helix C is displaced from its active state position. Finally, the structure provides for the first time a molecular rationale that explains how mutations in MEK may lead to the cardio-facio-cutaneous syndrome.

Fully activated MEK1 exhibits compromised affinity for binding of allosteric inhibitors U0126 and PD0325901.

Kinases catalyze the transfer of γ-phosphate from ATP to substrate protein residues triggering signaling pathways responsible for a plethora of cellular events. Isolation and production of homogeneous preparations of kinases in their fully active forms is important for accurate in vitro measurements of activity, stability, and ligand binding properties of these proteins. Previous studies have shown that MEK1 can be produced in its active phosphorylated form by coexpression with RAF1 in insect cells. In this study, using activated MEK1 produced by in vitro activation by RAF1 (pMEK1(in vitro)), we demonstrate that the simultaneous expression of RAF1 for production of activated MEK1 does not result in stoichiometric phosphorylation of MEK1. The pMEK1(in vitro) showed higher specific activity toward ERK2 protein substrate compared to the pMEK1 that was activated via coexpression with RAF1 (pMEK1(in situ)). The two pMEK1 preparations showed quantitative differences in the phosphorylation of T-loop residue serine 222 by Western blotting and mass spectrometry. Finally, pMEK1(in vitro) showed marked differences in the ligand binding properties compared to pMEK1(in situ). Contrary to previous findings, pMEK1(in vitro) bound allosteric inhibitors U0126 and PD0325901 with a significantly lower affinity than pMEK1(in situ) as well as its unphosphorylated counterpart (npMEK1) as demonstrated by thermal-shift, AS-MS, and calorimetric studies. The differences in inhibitor binding affinity provide direct evidence that unphosphorylated and RAF1-phosphorylated MEK1 form distinct inhibitor sites.

Development of a fluorescence polarization bead-based coupled assay to target different activity/conformation states of a protein kinase.

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP™)-based couple™d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z′ of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC™ and validated with the protein kinase C inhibitor staurosporine. The IC50 value generated was comparable to the value obtained by the radioactive 33P-γ-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.