The role of a novel auxiliary pocket in bacterial phenylalanyl-tRNA synthetase druggability.

The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.

Discovery of OICR12694: A Novel, Potent, Selective, and Orally Bioavailable BCL6 BTB Inhibitor.

B cell lymphoma 6 (BCL6), a highly regulated transcriptional repressor, is deregulated in several forms of non-Hodgkin lymphoma (NHL), most notably in diffuse large B-cell lymphoma (DLBCL). The activities of BCL6 are dependent on protein-protein interactions with transcriptional co-repressors. To find new therapeutic interventions addressing the needs of patients with DLBCL, we initiated a program to identify BCL6 inhibitors that interfere with co-repressor binding. A virtual screen hit with binding activity in the high micromolar range was optimized by structure-guided methods, resulting in a novel and highly potent inhibitor series. Further optimization resulted in the lead candidate 58 (OICR12694/JNJ-65234637), a BCL6 inhibitor with low nanomolar DLBCL cell growth inhibition and an excellent oral pharmacokinetic profile. Based on its overall favorable preclinical profile, OICR12694 is a highly potent, orally bioavailable candidate for testing BCL6 inhibition in DLBCL and other neoplasms, particularly in combination with other therapies.

Thienopyrrole acetic acids as antagonists of the CRTH2 receptor.

The bioisosteric replacement of the indole core of CRTH2 antagonists using thienopyrroles was investigated, resulting in potent antagonists with good selectivity over DP1. Early ADME/PK assessment of this chemotype demonstrated bioavailability in mice.

Design, Synthesis, and Characterization of 4-Aminoquinazolines as Potent Inhibitors of the G Protein-Coupled Receptor Kinase 6 (GRK6) for the Treatment of Multiple Myeloma.

Both previous and additional genetic knockdown studies reported herein implicate G protein-coupled receptor kinase 6 (GRK6) as a critical kinase required for the survival of multiple myeloma (MM) cells. Therefore, we sought to develop a small molecule GRK6 inhibitor as an MM therapeutic. From a focused library of known kinase inhibitors, we identified two hits with moderate biochemical potencies against GRK6. From these hits, we developed potent (IC50 < 10 nM) analogues with selectivity against off-target kinases. Further optimization led to the discovery of an analogue (18) with an IC50 value of 6 nM against GRK6 and selectivity against a panel of 85 kinases. Compound 18 has potent cellular target engagement and antiproliferative activity against MM cells and is synergistic with bortezomib. In summary, we demonstrate that targeting GRK6 with small molecule inhibitors represents a promising approach for MM and identify 18 as a novel, potent, and selective GRK6 inhibitor.

Discovery of A-971432, An Orally Bioavailable Selective Sphingosine-1-Phosphate Receptor 5 (S1P5) Agonist for the Potential Treatment of Neurodegenerative Disorders.

S1P5 is one of 5 receptors for sphingosine-1-phosphate and is highly expressed on endothelial cells within the blood-brain barrier, where it maintains barrier integrity in in vitro models (J. Neuroinflamm. 2012, 9, 133). Little more is known about the effects of S1P5 modulation due to the absence of tool molecules with suitable selectivity and drug-like properties. We recently reported that molecule A-971432 (Harris, 2010) (29 in this paper) is highly efficacious in reversing lipid accumulation and age-related cognitive decline in rats (Van der Kam , , AAIC 2014). Herein we describe the development of a series of selective S1P5 agonists that led to the identification of compound 29, which is highly selective for S1P5 and has excellent plasma and CNS exposure after oral dosing in preclinical species. To further support its suitability for in vivo studies of S1P5 biology, we extensively characterized 29, including confirmation of its selectivity in pharmacodynamic assays of S1P1 and S1P3 function in rats. In addition, we found that 29 improves blood-brain barrier integrity in an in vitro model and reverses age-related cognitive decline in mice. These results suggest that S1P5 agonism is an innovative approach with potential benefit in neurodegenerative disorders involving lipid imbalance and/or compromised blood-brain barrier such as Alzheimer's disease or multiple sclerosis.

Selective inhibitors of bacterial t-RNA-(N(1)G37) methyltransferase (TrmD) that demonstrate novel ordering of the lid domain.

The tRNA-(N(1)G37) methyltransferase (TrmD) is essential for growth and highly conserved in both Gram-positive and Gram-negative bacterial pathogens. Additionally, TrmD is very distinct from its human orthologue TRM5 and thus is a suitable target for the design of novel antibacterials. Screening of a collection of compound fragments using Haemophilus influenzae TrmD identified inhibitory, fused thieno-pyrimidones that were competitive with S-adenosylmethionine (SAM), the physiological methyl donor substrate. Guided by X-ray cocrystal structures, fragment 1 was elaborated into a nanomolar inhibitor of a broad range of Gram-negative TrmD isozymes. These compounds demonstrated no activity against representative human SAM utilizing enzymes, PRMT1 and SET7/9. This is the first report of selective, nanomolar inhibitors of TrmD with demonstrated ability to order the TrmD lid in the absence of tRNA.

Specific versus nonspecific binding of cationic PNAs to duplex DNA.

Although peptide nucleic acids (PNAs) are neutral by themselves, they are usually appended with positively charged lysine residues to increase their solubility and binding affinity for nucleic acid targets. Thus obtained cationic PNAs very effectively interact with the designated duplex DNA targets in a sequence-specific manner forming strand-invasion complexes. We report on the study of the nonspecific effects in the kinetics of formation of sequence-specific PNA-DNA complexes. We find that in a typical range of salt concentrations used when working with strand-invading PNAs (10-20 mM NaCl) the PNA binding rates essentially do not depend on the presence of nontarget DNA in the reaction mixture. However, at lower salt concentrations (<10 mM NaCl), the rates of PNA binding to DNA targets are significantly slowed down by the excess of unrelated DNA. This effect of nontarget DNA arises from depleting the concentration of free PNA capable of interacting with DNA target due to adhesion of positively charged PNA molecules on the negatively charged DNA duplex. As expected, the nonspecific electrostatic effects are more pronounced for more charged PNAs. We propose a simple model quantitatively describing all major features of the observed phenomenon. This understanding is important for design of and manipulation with the DNA-binding polycationic ligands in general and PNA-based drugs in particular.

CS-dependent response probability in an auditory masked-detection task: considerations based on models of Pavlovian conditioning.

Experimental studies were performed using a Pavlovian-conditioned eyeblink response to measure detection of a variable-sound-level tone (T) in a fixed-sound-level masking noise (N) in rabbits. Results showed an increase in the asymptotic probability of conditioned responses (CRs) to the reinforced TN trials and a decrease in the asymptotic rate of eyeblink responses to the non-reinforced N presentations as a function of the sound level of the T. These observations are consistent with expected behaviour in an auditory masked detection task, but they are not consistent with predictions from a traditional application of the Rescorla-Wagner or Pearce models of associative learning. To implement these models, one typically considers only the actual stimuli and reinforcement on each trial. We found that by considering perceptual interactions and concepts from signal detection theory, these models could predict the CS dependence on the sound level of the T. In these alternative implementations, the animals response probabilities were used as a guide in making assumptions about the "effective stimuli".