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.

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.

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 (EC 50 < 500 nM) and good selectivity over the activity of human HDAC in cells (up to >50-fold). Inhibition of parasital HDACs as the mechanism of action of this new class of selective growth inhibitors is supported by hyperacetylation studies.KEYWORDS: Malaria, Plasmodium falciparum, PfHDAC1, 4-arylimidazoles I nfection with malaria parasites such as Plasmodium falciparum remains a devastating cause of death in tropical geographies with 40% of the world population at risk of acquiring the disease. There are approximately 200 million clinical cases of malaria every year leading to an estimated 600,000 deaths.1 The requirement for improved therapies to treat and to cure malaria is an evident medical and humanitarian need that is exacerbated by an alarming rise in parasite resistance to the current standard of care.2,3 Drugs that operate via novel mechanisms of action for which no innately resistant parasites are expected are therefore especially desirable.DNA is tightly packed around histone proteins in the nucleus of eukaryotic cells with its transcription being regulated by chemical modifications to the nucleosomal histone proteins themselves. Histone deacetylases (HDACs) are zinc-dependent enzymes that play crucial roles in modulating mammalian cell chromatin structure, transcription, and gene expression. 4−6HDACs have also been identified as important regulators of transcription in P. falciparum, 7−10 and inhibition of P. falciparum histone deacetylases (Pf HDACs) has been reported to both effectively kill the parasites (Vorinostat, Figure 1) 11−16 and lead to efficacy in animal models of malaria (compound 2).17 Such findings underscore the potential for Pf HDAC inhibitors to be used for malaria therapy. 18−20 Of the five HDAC encoding genes known in P. falciparum one has homology to mammalian class I isoforms (Pf HDAC1), two are similar class II (Pf HDAC2 and 3) mammalian HDACs, while the remaining two are class III HDACs, or silent information regulator 2 (SIR2) proteins. 19 In light of the close sequence homology between Pf HDAC1 and human class I HDACs 21 an

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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. (Journal of Biomolecular Screening 2008:862-869)