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Chronic hepatitis B (CHB) is a major worldwide public health problem and novel anti-HBV therapies preventing liver disease progression to cirrhosis and hepatocellular carcinoma are urgently needed. Over the last several years, capsid assembly modulators (CAM) have emerged as clinically effective anti-HBV agents which can inhibit HBV replication in CHB patients. As part of a drug discovery program aimed at obtaining novel CAM endowed with high in vitro and in vivo antiviral activity, we identified a novel series of sulfamoylbenzamide (SBA) derivatives. Compound 10, one of the most in vitro potent SBA-derived CAM discovered to date, showed excellent pharmacokinetics in mice suitable for oral dosing. When studied in a transgenic mouse model of hepatic HBV replication, it was considerably more potent than NVR 3-778, the first sulfamoylbenzamide (SBA) CAM that entered clinical trials for CHB, at reducing viral replication in a dose-dependent fashion. We present herein the discovery process, the SAR analysis and the pre-clinical profile of this novel SBA CAM.

Coenzyme A (CoA) is a fundamental cofactor involved in a number of important biochemical reactions in the cell. Altered CoA metabolism results in severe conditions such as pantothenate kinase-associated neurodegeneration (PKAN) in which a reduction of the activity of pantothenate kinase isoform 2 (PANK2) present in CoA biosynthesis in the brain consequently lowers the level of CoA in this organ. In order to develop a new drug aimed at restoring the sufficient amount of CoA in the brain of PKAN patients, we looked at its turnover. We report here the results of two experiments that enabled us to measure the half-life of pantothenic acid, free CoA (CoASH) and acetylCoA in the brains and livers of male and female C57BL/6N mice, and total CoA in the brains of male mice. We administered (intrastriatally or orally) a single dose of a [13C3-15N-18O]-labelled coenzyme A precursor (fosmetpantotenate or [13C3-15N]-pantothenic acid) to the mice and measured, by liquid chromatography-mass spectrometry, unlabelled- and labelled-coenzyme A species appearance and disappearance over time. We found that the turnover of all metabolites was faster in the liver than in the brain in both genders with no evident gender difference observed. In the oral study, the CoASH half-life was: 69 ± 5 h (male) and 82 ± 6 h (female) in the liver; 136 ± 14 h (male) and 144 ± 12 h (female) in the brain. AcetylCoA half-life was 74 ± 9 h (male) and 71 ± 7 h (female) in the liver; 117 ± 13 h (male) and 158 ± 23 (female) in the brain. These results were in accordance with the corresponding values obtained after intrastriatal infusion of labelled-fosmetpantotenate (CoASH 124 ± 13 h, acetylCoA 117 ± 11 and total CoA 144 ± 17 in male brain).

A previous publication from our laboratory reported the identification of a new class of 2-(1H-imidazo-2-yl)piperazines as potent T. brucei growth inhibitors as potential treatment for Human African Trypanosomiasis (HAT). This work describes the structure–activity relationship (SAR) around the hit compound 1, which led to the identification of the optimized compound 18, a single digit nanomolar inhibitor (EC50 7 nM), not cytotoxic and with optimal in vivo profile that made it a suitable candidate for efficacy studies in a mouse model mimicking the second stage of disease.

Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are associated with neurodegenerative disorders. Tryptophan is transported across the blood–brain barrier and converted via the kynurenine pathway to N-formyl- l -kynurenine, which is further degraded to l -kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties. The association of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in analytical methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC–MS/MS method to quantify l -kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy- l -kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chemical properties, major efforts were devoted to develop a chromatography suitable for all metabolites that involves plasma protein precipitation with acetonitrile followed by chromatographic separation by C18 RP chromatography, detected by electrospray mass spectrometry. Quantitation range was 0.098–100 ng/ml for 3HK, 9.8–20,000 ng/ml for KYN, 0.49–1000 ng/ml for KYNA and AA. The method was linear (r > 0.9963) and validation parameters were within acceptance range (calibration standards and QC accuracy within ±30%).

The identification of a new series of P. falciparum growth inhibitors is described. Starting from a series of known human class I HDAC inhibitors a SAR exploration based on growth inhibitory activity in parasite and human cells-based assays led to the identification of compounds with submicromolar inhibition of P. falciparum growth (EC50500 nM) and good selectivity over the activity of human HDAC in cells (up to50-fold). Inhibition of parasital HDACs as the mechanism of action of this new class of selective growth inhibitors is supported by hyperacetylation studies.

The Azotobacter vinelandii rhodanese is a 31kDa sulfurtransferase protein that catalyzes the transfer of sulfur atom from thiosulfate to cyanide in the detoxification process from cyanide and is able to insert sulfur atom in the iron-sulfur cluster. A study of the uniformly 15N isotopic labeling by high resolution NMR, before obtaining the backbone sequential assignment, has been carried out. The sulfur loaded and the sulfur discharged forms of the enzyme show very similar HSQC spectra with a good spectral dispersion. Few resonances show changes in chemical shift between the two forms. Relaxation parameters T(1), T(2) and 1H-15N NOE of all amide nitrogen atoms, as well as isotope exchange kinetics, show that the two forms exhibit the same global correlation time and hydrodynamic properties. In parallel, essential dynamics studies show that formation and discharging of catalytic cysteine persulfide group has no significant impact on the overall conformation of the protein. These results, taken together, give a clearcut answer to the question if the catalytic mechanism of the enzyme involves a change in the conformation and/or in the mutual orientation of the two domains. On the contrary these results clearly indicate that upon the catalytic mechanism the two domains of the protein behave as a unique fold.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.

We disclose the development of a novel series of 2-phenyl-2H-indazole-7-carboxamides as poly(ADP-ribose)polymerase (PARP) 1 and 2 inhibitors. This series was optimized to improve enzyme and cellular activity, and the resulting PARP inhibitors display antiproliferation activities against BRCA-1 and BRCA-2 deficient cancer cells, with high selectivity over BRCA proficient cells. Extrahepatic oxidation by CYP450 1A1 and 1A2 was identified as a metabolic concern, and strategies to improve pharmacokinetic properties are reported. These efforts culminated in the identification of 2-{4-[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide 56 (MK-4827), which displays good pharmacokinetic properties and is currently in phase I clinical trials. This compound displays excellent PARP 1 and 2 inhibition with IC(50) = 3.8 and 2.1 nM, respectively, and in a whole cell assay, it inhibited PARP activity with EC(50) = 4 nM and inhibited proliferation of cancer cells with mutant BRCA-1 and BRCA-2 with CC(50) in the 10-100 nM range. Compound 56 was well tolerated in vivo and demonstrated efficacy as a single agent in a xenograft model of BRCA-1 deficient cancer.

In this work, the authors present a novel, robotic, automated protocol for assessing a metabolic stability protocol assembled on a Hamilton platform and a new strategy for pooling samples (cassette analysis). To increase the high throughput of the liquid chromatography (LC) step, fast chromatography and automated liquid chromatography tandem mass spectrometry (LC/MS/MS) analytical methods were also developed, and a rapid data analysis system was generated that converts peak areas obtained by LC/MS/MS in intrinsic clearance values. All of the steps of the microsomal stability assay were carefully studied and optimized. Standard errors and confidence intervals of the measured clearances were also automatically generated in the process to allow an immediate evaluation of the significance of observed values. Methods based on pooling analysis of 2 and 4 different analytes were compared with a standard method without pooling. A simple statistical treatment was used to show their equivalence. The different protocols developed were analyzed in terms of the best compromise between accuracy and high-throughput capabilities.

Trifluoroacetylthiophene carboxamides have recently been reported to be class II HDAC inhibitors, with moderate selectivity. Exploration of replacements for the carboxamide with bioisosteric pentatomic heteroaromatic like 1,3,4-oxadiazoles, 1,2,4-oxadiazoles and 1,3-thiazoles, led to the discovery that 2-trifluoroacetylthiophene 1,3,4-oxadiazole derivatives are very potent low nanomolar HDAC4 inhibitors, highly selective over class I HDACs (HDAC 1 and 3), and moderately stable in HCT116 cell culture.

The NMR high-resolution structure of calmodulin complexed with a fragment of the olfactory cyclic-nucleotide gated channel is described. This structure shows features that are unique for this complex, including an active role of the linker connecting the N- and C-lobes of calmodulin upon binding of the peptide. Such linker is not only involved in the formation of an hydrophobic pocket to accommodate a bulky peptide residue, but it also provides a positively charged region complementary to a negative charge of the target. This complex of calmodulin with a target not belonging to the kinase family was used to test the residual dipolar coupling (RDC) approach for the determination of calmodulin binding modes to peptides. Although the complex here characterized belongs to the (1--14) family, high Q values were obtained with all the 1:1 complexes for which crystalline structures are available. Reduction of the RDC data set used for the correlation analysis to structured regions of the complex allowed a clear identification of the binding mode. Excluded regions comprise calcium binding loops and loops connecting the EF-hand motifs.

Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [15N]Cys, and changes in 1H and 15N NMR resonances on addition of different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions. Moreover, the 1H-NMR spectrum of a defined spectral region is indicative of the status of the enzyme and can be used to directly monitor sulfur loading even at low concentrations. Selenium loading by the addition of selenodiglutathione was monitored by fluorescence and NMR spectroscopy. It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme. These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo.