Potent, selective and orally bioavailable dihydropyrimidine inhibitors of Rho kinase (ROCK1) as potential therapeutic agents for cardiovascular diseases.

Recent studies using known Rho-associated kinase isoform 1 (ROCK1) inhibitors along with cellular and molecular biology data have revealed a pivotal role of this enzyme in many aspects of cardiovascular function. Here we report a series of ROCK1 inhibitors which were originally derived from a dihydropyrimidinone core 1. Our efforts focused on the optimization of dihydropyrimidine 2, which resulted in the identification of a series of dihydropyrimidines with improved pharmacokinetics and P450 properties.

Discovery of aminofurazan-azabenzimidazoles as inhibitors of Rho-kinase with high kinase selectivity and antihypertensive activity.

The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.

Development of dihydropyridone indazole amides as selective Rho-kinase inhibitors.

Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.

Synthesis and characterization of 5'-p-fluorosulfonylbenzoyl-2' (or 3')-(biotinyl)adenosine as an activity-based probe for protein kinases.

Most of the kinase inhibitors that are approved for therapeutic uses or that are undergoing clinical trials are directed toward the adenosine triphosphate (ATP) binding site of protein kinases. 5'-Fluorosulfonylbenzoyl 5'-adenosine (FSBA) is an activitybased probe (ABP) that covalently modifies a conserved lysine present in the nucleotide binding site of most kinases. Here the authors describe synthesis of FSBA derivatives, 2'-biotinyl-FSBA and 3'-biotinyl-FSBA as kinase ABPs, and delineate a Western blot method to screen and validate ATP competitive protein kinase inhibitors using biotinyl-FSBA as a nonselective activity-based probe for protein kinases.

Expression, purification, and characterization of an enzymatically active truncated human rho-kinase I (ROCK I) domain expressed in Sf-9 insect cells.

Rho Kinase I (ROCK I) is a serine/threonine kinase that is involved in diverse cellular signaling. To further understand the physiological role of ROCK I and to identify and develop potent and selective inhibitors of ROCK I, we have overexpressed and purified a constitutively active dimeric human ROCK I (3-543) kinase domain using the Sf9-baculovirus expression system. In addition, using a limited proteolysis technique, we have identified a minimal functional subdomain of ROCK I that can be used in crystallization studies. The availability of multimilligram amounts of purified and well characterized functional human ROCK I kinase domains will be useful in screening and structural studies.

A liquid chromatography/mass spectrometry-based method for the selection of ATP competitive kinase inhibitors.

The currently approved kinase inhibitors for therapeutic uses and a number of kinase inhibitors that are undergoing clinical trials are directed toward the adenosine triphosphate (ATP) binding site of protein kinases. The 5'-fluorosulfonylbenzoyl 5'-adenosine (FSBA) is an ATP-affinity reagent that covalently modifies a conserved lysine present in the nucleotide-binding site of most kinases. The authors have developed a liquid chromatography/mass spectrometry-based method to monitor binding of ATP competitive protein kinase inhibitors using FSBA as a nonselective activity-based probe for protein kinases. Their method provides a general, rapid, and reproducible means to screen and validate selective ATP competitive inhibitors of protein kinases.

Crystal structure and substrate specificity of the beta-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus.

beta-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 A resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rank order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.

The RNA-unwinding activity of hepatitis C virus non-structural protein 3 (NS3) is positively modulated by its protease domain.

The nonstructural protein 3 (NS3) of hepatitis C virus contains a protease domain at its amino terminus and RNA helicase domain at its carboxyl terminus. To identify optimal NS3 protein for developing screening assays, we expressed full-length NS3 protease/helicase and helicase domains from both HCV type 1a (H77 strain) and 1b (Con1 strain), using either E. coli or baculovirus expression systems. Our studies showed that the full-length NS3 proteins, either with or without the presence of the NS4A domain, from either strains were at least 10-fold more efficient than the corresponding helicase domains in unwinding partial duplex RNA substrates. These findings provide a rationale for the use of full-length NS3 in high throughput screening assays to identify potent small molecule inhibitors of this important target of HCV.

Bacterial beta-ketoacyl-acyl carrier protein synthases as targets for antibacterial agents.

As a result of increasing drug resistance in pathogenic bacteria, there is a critical need for novel broad-spectrum antibacterial agents. As fatty acid synthesis (FAS) in bacteria is an essential process for cell survival, the enzymes involved in the FAS pathway have emerged as promising targets for antimicrobial agents. Several lines of evidence have indicated that bacterial condensing enzymes are central to the initiation and elongation steps in bacterial fatty acid synthesis and play a pivotal role in the regulation of the entire fatty acid synthesis pathway. beta-ketoacyl-acyl carrier protein (ACP) synthases (KAS) from various bacterial species have been cloned, expressed and purified in large quantities for detailed enzymological, structural and screening studies. Availability of purified KAS from a variety of bacteria, along with a combination of techniques, including combinatorial chemistry, high-throughput screening, and rational drug design based on crystal structures, will undoubtedly aid in the discovery and development of much needed potent and broad-spectrum antibacterial agents. In this review we summarize the biochemical, biophysical and inhibition properties of beta-ketoacyl-ACP synthases from a variety of bacterial species.

First X-ray cocrystal structure of a bacterial FabH condensing enzyme and a small molecule inhibitor achieved using rational design and homology modeling.

The first cocrystal structure of a bacterial FabH condensing enzyme and a small molecule inhibitor is reported. The inhibitor was obtained by rational modification of a high throughput screening lead with the aid of a S. pneumoniae FabH homology model. This homology model was used to design analogues that would have both high affinity for the enzyme and appropriate aqueous solubility to facilitate cocrystallization studies.