Identification of Cosalane as an Inhibitor of Human and Murine CC-Chemokine Receptor 7 Signaling via a High-Throughput Screen.

CC-chemokine receptor 7 (CCR7) is a G protein-coupled receptor expressed on a variety of immune cells. CCR7 plays a critical role in the migration of lymphocytes into secondary lymphoid tissues. CCR7 expression, however, has been linked to numerous disease states. Due to its therapeutic relevance and absence of available CCR7 inhibitors, we undertook a high-throughput screen (HTS) to identify small-molecule antagonists of the receptor. Here, we describe a robust HTS approach using a commercially available ß-galactosidase enzyme fragment complementation system and confirmatory transwell chemotaxis assays. This work resulted in the identification of several compounds with activity against CCR7. The most potent of these was subsequently determined to be cosalane, a cholesterol derivative previously designed as a therapeutic for human immunodeficiency virus. Cosalane inhibited both human and murine CCR7 in response to both CCL19 and CCL21 agonists at physiologic concentrations. Furthermore, cosalane produced durable inhibition of the receptor following a cellular incubation period with subsequent washout. Overall, our work describes the development of an HTS-compatible assay, completion of a large HTS campaign, and demonstration for the first time that cosalane is a validated CCR7 antagonist. These efforts could pave the way for new approaches to address CCR7-associated disease processes.

Filtration improves the performance of a high-throughput screen for anti-mycobacterial compounds.

The tendency for mycobacteria to aggregate poses a challenge for their use in microplate based assays. Good dispersions have been difficult to achieve in high-throughput screening (HTS) assays used in the search for novel antibacterial drugs to treat tuberculosis and other related diseases. Here we describe a method using filtration to overcome the problem of variability resulting from aggregation of mycobacteria. This method consistently yielded higher reproducibility and lower variability than conventional methods, such as settling under gravity and vortexing.

Development of a high-throughput assay for identifying inhibitors of TBK1 and IKKε.

IKKε and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKε has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKε. This information enabled the design of an optimal TBK1/IKKε substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKε. 227 compounds in this library inhibited TBK1 at a concentration of 10 µM, while 57 compounds inhibited IKKε. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKε inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.

Inhibition of NFkappaB by the natural product Withaferin A in cellular models of Cystic Fibrosis inflammation.

Cystic Fibrosis (CF) is one of the most common autosomal genetic disorders in humans. This disease is caused by mutations within a single gene, coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The phenotypic hallmark of CF is chronic lung infection and associated inflammation from opportunistic microbes such as Pseudomonas aeruginosa (PA), Haemophilus influenzae, and Staphylococcus aureus. This eventually leads to deterioration of lung function and death in most CF patients. Unfortunately, there is no approved therapy for correcting the genetic defect causal to the disease. Hence, controlling inflammation and infection in CF patients are critical to disease management. Accordingly, anti-inflammatory agents and antibiotics are used to manage chronic inflammation and infection in CF patients. However, most of the anti-inflammatory agents in CF have severe limitations due to adverse side effects, and resistance to antibiotics is becoming an even more prominent problem. Thus, new agents that can be used to control chronic inflammation in CF are needed in the absence of a cure for the disease. Activation of the transcription factor NFkappaB through Toll-like receptors (TLR) following bacterial infection is principally involved in regulating lung inflammation in CF. NFkappaB regulates the transcription of several genes that are involved in inflammation, anti-apoptosis and anti-microbial activity, and hyper-activation of this transcription factor leads to a potent inflammatory response. Thus, NFkappaB is a potential anti-inflammatory drug target in CF. Screening of several compounds from natural sources in an in vitro model of CF-related inflammation wherein NFkappaB is activated by filtrates of a clinically isolated strain of PA (PAF) led us to Withaferin A (WFA), a steroidal lactone from the plant Withania Somnifera L. Dunal. Our data demonstrate that WFA blocks PAF-induced activation of NFkappaB as determined using reporter assays, IL-8 measurements and high-content fluorescent imaging of NFkappaB subunit p65 translocation. Since the airways of CF patients can be specifically targeted for delivery of therapeutics, we propose that WFA should be further studied as an anti-inflammatory agent in models of CF related inflammation mediated by NFkappaB.

Hydrogen bonding is the prime determinant of carboxyl pKa values at the N-termini of alpha-helices.

Experimentally determined mean pK(a) values of carboxyl residues located at the N-termini of alpha-helices are lower than their overall mean values. Here, we perform three types of analyses to account for this phenomenon. We estimate the magnitude of the helix macrodipole to determine its potential role in lowering carboxyl pK(a) values at the N-termini. No correlation between the magnitude of the macrodipole and the pK(a) values is observed. Using the pK(a) program propKa we compare the molecular surroundings of 18 N-termini carboxyl residues versus 233 protein carboxyl groups from a previously studied database. Although pK(a) lowering interactions at the N-termini are similar in nature to those encountered in other protein regions, pK(a) lowering backbone and side-chain hydrogen bonds appear in greater number at the N-termini. For both Asp and Glu, there are about 0.5 more hydrogen bonds per residue at the N-termini than in other protein regions, which can be used to explain their lower than average pK(a) values. Using a QM-based pK(a) prediction model, we investigate the chemical environment of the two lowest Asp and the two lowest Glu pK(a) values at the N-termini so as to quantify the effect of various pK(a) determinants. We show that local interactions suffice to account for the acidity of carboxyl residues at the N-termini. The effect of the helix dipole on carboxyl pK(a) values, if any, is marginal. Backbone amide hydrogen bonds constitute the single biggest contributor to the lowest carboxyl pK(a) values at the N-termini. Their estimated pK(a) lowering effects range from about 1.0 to 1.9 pK(a) units.

The Low-Barrier Double-Well Potential of the O(δ)(1)-H-O(δ)(1) Hydrogen Bond in Unbound HIV Protease: A QM/MM Characterization.

The presence of a low-barrier hydrogen bond (LBHB) in aspartyl proteases and its implications in drug design have been the subject of intense study. Here, we present a combined quantum mechanical/molecular mechanical (QM/MM)-Numerov procedure and use it to characterize the O(δ)(1)-H-O(δ)(1) hydrogen bond (HB) in unbound HIV protease. The QM/MM scheme fully traces the shape of the HB's potential energy curve. The potential is used to obtain numerical solutions to the wave functions and vibrational energies of hydrogen, deuterium, and tritium. The vibrational eigenfunctions are used to compute expectation values for interatomic distances and vibrationally and thermally averaged spectroscopic properties of the O(δ)(1)-H-O(δ)(1) HB. Our work corroborates previous results by Piana and Carloni who found a LBHB via an ab initio molecular dynamics simulation (Piana, S.; Carloni, P. Proteins 2000, 39, 26-36). Our predictions of isotope effects on the chemical shift of unbound HIV protease are consistent with experimental measurements in similar HBs. These results support the predictive power of this method and its potential use in screening inhibitors of aspartyl proteases.