Identification of Selective Dual ROCK1 and ROCK2 Inhibitors Using Structure-Based Drug Design.

A HTS campaign identified compound 1, an excellent hit-like molecule to initiate medicinal chemistry efforts to optimize a dual ROCK1 and ROCK2 inhibitor. Substitution (2-Cl, 2-NH2, 2-F, 3-F) of the pyridine hinge binding motif or replacement with pyrimidine afforded compounds with a clean CYP inhibition profile. Cocrystal structures of an early lead compound were obtained in PKA, ROCK1, and ROCK2. This provided critical structural information for medicinal chemistry to drive compound design. The structural data indicated the preferred configuration at the central benzylic carbon would be ( R), and application of this information to compound design resulted in compound 16. This compound was shown to be a potent and selective dual ROCK inhibitor in both enzyme and cell assays and efficacious in the retinal nerve fiber layer model after oral dosing. This tool compound has been made available through the AbbVie Compound Toolbox. Finally, the cocrystal structures also identified that aspartic acid residues 176 and 218 in ROCK2, which are glutamic acids in PKA, could be targeted as residues to drive both potency and kinome selectivity. Introduction of a piperidin-3-ylmethanamine group to the compound series resulted in compound 58, a potent and selective dual ROCK inhibitor with excellent predicted drug-like properties.

Design of Aminobenzothiazole Inhibitors of Rho Kinases 1 and 2 by Using Protein Kinase†A as a Structure Surrogate.

We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinase A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model.

Fragment-based discovery of a potent NAMPT inhibitor.

NAMPT expression is elevated in many cancers, making this protein a potential target for anticancer therapy. We have carried out both NMR based and TR-FRET based fragment screens against human NAMPT and identified six novel binders with a range of potencies. Co-crystal structures were obtained for two of the fragments bound to NAMPT while for the other four fragments force-field driven docking was employed to generate a bound pose. Based on structural insights arising from comparison of the bound fragment poses to that of bound FK866 we were able to synthetically elaborate one of the fragments into a potent NAMPT inhibitor.

Implementation of a human podocyte injury model of chronic kidney disease for profiling of renoprotective compounds.

Degradation of podocyte structural integrity and function are hallmarks of proteinuric chronic kidney disease. In vivo, injury of podocytes manifests itself in the form of disruption of foot process morphology and associated cytoskeletal architecture, de-differentiation, and loss of adhesion to the glomerular basement membrane. Given the critical role played by this highly specialized cell type in maintaining glomerular filtration, there is a need for improved physiologically relevant cellular models that enable detection of disease-relevant indicators of podocyte perturbation. We have addressed this need by evaluating a subclone of conditionally immortalized human podocytes through quantitative benchmarking against freshly isolated primary human podocytes. Benchmarking was performed by measuring key phenotypic parameters, expression of podocyte specific proteins and multiparametric responses to stressors that model different aspects of podocyte perturbation. We subsequently employed the subcloned cells to profile the protective activity of structurally distinct adenosine kinase inhibitors. Our results support the translatability of our cellular model and set the stage for broader screening of renoprotective compounds with a view to eventually treat proteinuric kidney disease.

A-306989, an inhibitor of adenosine kinase, is renoprotective in rodent models of podocyte, basement membrane, and obstructive injury.

Adenosine (ADO) is an important regulatory purine nucleoside that accumulates at sites of inflammation and tissue injury including in diseases associated with renal pathology. Endogenous levels of ADO may be increased by inhibiting the ADO-metabolizing enzyme, ADO kinase (AK). AK inhibitors have demonstrated protection in rodent models of diabetic nephropathy. To further investigate AK inhibition as a potential mechanism for renal protection, A-306989, a potent non-nucleoside AK inhibitor, was examined in both in vitro and in vivo assays of renal injury. A-306989 prevented podocyte damage (disruption of actin cytoskeleton) and increased podocyte survival following puromycin aminonucleoside (PAN) application in both mouse and human conditionally immortalized podocytes. Prophylactic oral administration of A-306989 (1.5, 5 and 15mg/kg) reduced proteinuria in a dose-dependent manner and repressed pro-inflammatory/fibrotic gene up-regulation; A-306989 was also efficacious when administered two days following the PAN-insult. A-306989 (10 and 30mg/kg) also significantly reduced proteinuria and macrophage infiltration in a rat model of glomerulonephritis. Finally, A-306989 (15 and 50mg/kg) reduced the expression levels of pro-inflammatory/fibrotic genes, and reduced macrophage infiltration (50mg/kg), but did not affect the deposition of interstitial collagen in fibrotic kidneys from mice with unilateral ureter obstruction. A-306989 also had beneficial actions on "quality of life" measures including improving body weight loss. Thus, these data indicate that enhancement of endogenous ADO levels by A-306989 can positively modulate renal pathology and mimic some of the previously reported beneficial actions of ADO A2A receptor agonists.

Development and validation of a fluorescence-based HTS assay for the identification of P/Q-type calcium channel blockers.

Dysfunction of P/Q-type calcium channels is thought to underlie a variety of neurological diseases. There is evidence that migraine, Alzheimer's disease, and epilepsy involve a gain-of-function of the channel, leading to abnormal presynaptic vesicle release. P/Q-channel blockers may normalize current flow and consequently lead to an alleviation of disease symptoms. Although the medical need is high, there are no such compounds on the market. Here we describe a high throughput screen (HTS) for P/Q-type calcium channel blockers and the confirmation of hits by automated electrophysiology. We generated a HEK293 cell line stably expressing the α1A subunit of the P/Q-type calcium channel under control of a tetracycline (Tet) promoter. The accessory ß1.1 and α2δ1 subunits were co-expressed constitutively. The cell line was pharmacologically characterized by ion channel specific modulators, and revealed functional P/Q-type calcium currents. Using a fluorescence imaging plate reader (FLIPR), an assay for P/Q-type calcium channels was established based on a calcium sensitive dye. HTS of a 150,000 compound-containing sub-library led to the identification of 3262 hits that inhibited the fluorescence signal with potencies below 10 µM. Hit-to-lead (HTL) efforts identified 12,400 analogues. Compounds were clustered into 37 series, and 8 series of interest were prioritized. An electrophysiological secondary screen, providing a more direct measure of channel function, was implemented into the HTL process. 27 selected exemplars of different chemotypes were validated by automated whole-cell patch clamp analysis at inactivated channel state. The discovery of P/Q-channel blockers may foster the development of new therapeutics for a variety of neurological diseases.

Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists.

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.

Novel antibacterial class: a series of tetracyclic derivatives.

We describe the synthesis and antibacterial activity of a series of tetracyclic naphthyridones. The members of this series act primarily via inhibition of bacterial translation and belong to the class of novel ribosome inhibitors (NRIs). In this paper we explore the structure-activity relationships (SAR) of these compounds to measure their ability both to inhibit bacterial translation and also to inhibit the growth of bacterial cells in culture. The most active of these compounds inhibit Streptococcus pneumoniae translation at concentrations of <5 microM and have minimum inhibitory concentrations (MICs) of <8 microg/mL against clinically relevant strains of bacteria.

Novel inhibitors of bacterial protein synthesis: structure-activity relationships for 1,8-naphthyridine derivatives incorporating position 3 and 4 variants.

Structure-activity relationships for a recently discovered novel ribosome inhibitor (NRI) class of antibacterials were investigated. Preliminary efforts to optimize protein synthesis inhibitory activity of the series through modification of positions 3 and 4 of the naphthyridone lead template resulted in the identification of several biochemically potent analogues. A lack of corresponding whole cell antibacterial activity is thought to be a consequence of poor cellular penetration as evidenced by the enhancement of activity observed for a lead analogue tested in the presence of a cell permeabilizing agent.

Novel antibacterial class.

We report the discovery and characterization of a novel ribosome inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial activity. Compounds in this class inhibit growth of many gram-positive and gram-negative bacteria, including the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis, and are nontoxic to human cell lines. The first NRI was discovered in a high-throughput screen designed to identify inhibitors of cell-free translation in extracts from S. pneumoniae. The chemical structure of the NRI class is related to antibacterial quinolones, but, interestingly, the differences in structure are sufficient to completely alter the biochemical and intracellular mechanisms of action. Expression array studies and analysis of NRI-resistant mutants confirm this difference in intracellular mechanism and provide evidence that the NRIs inhibit bacterial protein synthesis by inhibiting ribosomes. Furthermore, compounds in the NRI series appear to inhibit bacterial ribosomes by a new mechanism, because NRI-resistant strains are not cross-resistant to other ribosome inhibitors, such as macrolides, chloramphenicol, tetracycline, aminoglycosides, or oxazolidinones. The NRIs are a promising new antibacterial class with activity against all major drug-resistant respiratory pathogens.