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.

Proprotein convertase subtilisin kexin-9 (PCSK9) is an important pharmacological target for decreasing low-density lipoprotein (LDL) in cardiovascular disease, although seemingly inaccessible to small molecule approaches. Compared with therapeutic IgG antibodies currently in development, targeting circulating PCSK9 with smaller molecular scaffolds could offer different profiles and reduced dose burdens. This inspired genesis of PCSK9-binding Adnectins, a protein family derived from human fibronectin-10th-type III-domain and engineered for high-affinity target binding. BMS-962476, an ∼11-kDa polypeptide conjugated to polyethylene glycol to enhance pharmacokinetics, binds with subnanomolar affinity to human. The X-ray cocrystal structure of PCSK9 with a progenitor Adnectin shows ∼910 Å 2 of PCSK9 surface covered next to the LDL receptor binding site, largely by residues of a single loop of the Adnectin. In hypercholesterolemic, overexpressing human PCSK9 transgenic mice, BMS-962476 rapidly lowered cholesterol and free PCSK9 levels. In genomic transgenic mice, BMS-962476 potently reduced free human PCSK9 (ED 50 ∼0.01 mg/kg) followed by ∼2-fold increases in total PCSK9 before return to baseline. Treatment of cynomolgus monkeys with BMS-962476 rapidly suppressed free PCSK9 .99% and LDL-cholesterol ∼55% with subsequent 6-fold increase in total PCSK9, suggesting reduced clearance of circulating complex. Liver sterol response genes were consequently downregulated, following which LDL and total PCSK9 returned to baseline. These studies highlight the rapid dynamics of PCSK9 control over LDL and liver cholesterol metabolism and characterize BMS-962476 as a potent and efficacious PCSK9 inhibitor.

Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus. In this study we showed through genetic disruption experiments that mvaA, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S. aureus. Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The mvaA gene was cloned from S. aureus DNA and expressed with an N-terminal His tag in Escherichia coli. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the S. aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a K i of 320 M, over 10 4 higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.

The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contribution to the binding energy arises from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets [the numbering of the subsites is as defined in Berger, A.; Schechter, I. Philos. Trans. R. Soc. London, Ser. B 1970, 257, 249-264]. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease that is currently in clinical trials.

Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine kinase involved in the phosphorylation of MAP proteins that regulate microtubule dynamics. Abnormal activity of MARK4 has been proposed to contribute to neurofibrillary tangle formation in Alzheimer's disease. The crystal structure of the catalytic and ubiquitin-associated domains of MARK4 with a potent pyrazolopyrimidine inhibitor has been determined to 2.8 Å resolution with an Rwork of 22.8%. The overall structure of MARK4 is similar to those of the other known MARK isoforms. The inhibitor is located in the ATP-binding site, with the pyrazolopyrimidine group interacting with the inter-lobe hinge region while the aminocyclohexane moiety interacts with the catalytic loop and the DFG motif, forcing the activation loop out of the ATP-binding pocket.