Plasmodial Kinase Inhibitors: License to Cure?

Advances in the genetics, function, and stage-specificity of Plasmodium kinases has driven robust efforts to identify targets for the design of antimalarial therapies. Reverse genomics following phenotypic screening against Plasmodia or related parasites has uncovered vulnerable kinase targets including PI4K, PKG, and GSK-3, an approach bolstered by access to human disease-directed kinase libraries. Alternatively, screening compound libraries against Plasmodium kinases has successfully led to inhibitors with antiplasmodial activity. As with other therapeutic areas, optimizing compound ADMET and PK properties in parallel with target inhibitory potency and whole cell activity becomes paramount toward advancing compounds as clinical candidates. These and other considerations will be discussed in the context of progress achieved toward deriving important, novel mode-of-action kinase-inhibiting antimalarial medicines.

Novel Antitubercular 6-Dialkylaminopyrimidine Carboxamides from Phenotypic Whole-Cell High Throughput Screening of a SoftFocus Library: Structure-Activity Relationship and Target Identification Studies.

A BioFocus DPI SoftFocus library of ∼35 000 compounds was screened against Mycobacterium tuberculosis (Mtb) in order to identify novel hits with antitubercular activity. The hits were evaluated in biology triage assays to exclude compounds suggested to function via frequently encountered promiscuous mechanisms of action including inhibition of the QcrB subunit of the cytochrome bc1 complex, disruption of cell-wall homeostasis, and DNA damage. Among the hits that passed this screening cascade, a 6-dialkylaminopyrimidine carboxamide series was prioritized for hit to lead optimization. Compounds from this series were active against clinical Mtb strains, while no cross-resistance to conventional antituberculosis drugs was observed. This suggested a novel mechanism of action, which was confirmed by chemoproteomic analysis leading to the identification of BCG_3193 and BCG_3827 as putative targets of the series with unknown function. Initial structure-activity relationship studies have resulted in compounds with moderate to potent antitubercular activity and improved physicochemical properties.

Pyrrolo[3,4-c]pyridine-1,3(2H)-diones: A Novel Antimycobacterial Class Targeting Mycobacterial Respiration.

High-throughput screening of a library of small polar molecules against Mycobacterium tuberculosis led to the identification of a phthalimide-containing ester hit compound (1), which was optimized for metabolic stability by replacing the ester moiety with a methyl oxadiazole bioisostere. A route utilizing polymer-supported reagents was designed and executed to explore structure-activity relationships with respect to the N-benzyl substituent, leading to compounds with nanomolar activity. The frontrunner compound (5h) from these studies was well tolerated in mice. A M. tuberculosis cytochrome bd oxidase deletion mutant (ΔcydKO) was hyper-susceptible to compounds from this series, and a strain carrying a single point mutation in qcrB, the gene encoding a subunit of the menaquinol cytochrome c oxidoreductase, was resistant to compounds in this series. In combination, these observations indicate that this novel class of antimycobacterial compounds inhibits the cytochrome bc1 complex, a validated drug target in M. tuberculosis.

Gold(III) complexes of pyridyl- and isoquinolylamido ligands: structural, spectroscopic, and biological studies of a new class of dual topoisomerase I and II inhibitors.

The structures, spectroscopy, and cytotoxicity of four novel nominally square-planar gold(III) chelates 1-4 with the general formula cis-AuCl2(X), where the ligand X is an anionic bidentate pyridyl- or isoquinolylamido chelating agent, are described. The Au-N(amido), Au-N(pyridyl), and Au-N(isoquinolyl) distances are 2.002(9)-2.016(3), 2.01(1)-2.037(3), and 2.037(3) Å, respectively. Density functional theory simulations afforded accurate gold(III) coordination geometries for 1-4 (bond distances and angles to within 5% of the X-ray values), while accurate transition energies were limited to those calculated in the UV spectral region. The complexes had variable stability in dimethyl sulfoxide: compound 3 (relatively rigid) was indefinitely stable, compounds 1 and 2 (conformationally flexible) slowly demetalated over 30 days, and 4 (extensively aromatic) formed an insoluble precipitate after 10 days (72 h in an aqueous buffer). The isoquinolylamido derivative 4 was sufficiently cytotoxic in the NCI-60 screen to undergo full five-dose testing. Notably low GI50 (1.8, 2.3, and 3.2 µM) and IC50 (4.0, 9.8, and 15 µM) values were recorded for the OVCAR-3, IGROV1, and SW-620 cell lines, respectively. Hierarchical cluster analysis employing the National Cancer Institute (NCI) data for known anticancer drugs and 4 revealed that compound 4 is mechanistically identical with the topoisomerase IIα (Top2) poison zorubicin and statistically similar to the topoisomerase IB (Top1) poisons camptothecin and 9-methoxycamptothecin. The Top2-catalyzed decatenation reaction of kinetoplast DNA was studied as a function of the concentration of 4: the compound acts as an interfacial poison of Top2 at low concentrations (<1 µM) and a catalytic inhibitor of the enzyme above 5 µM. Gel mobility shift assays (plasmid DNA substrate) showed that the catalytic inhibition of Top2 likely correlates with DNA binding by 4 at concentrations >5 µM. Compound 4 is also a catalytic inhibitor of Top1 at higher concentrations, consistent with DNA binding by the complex.

Unconventional hydrogen bonding and π-stacking in two substituted pyridine carboxamides.

The crystal structures of two para-substituted aryl derivatives of pyridine-2-carboxamide, namely N-(4-fluorophenyl)pyridine-2-carboxamide, C(12)H(9)FN(2)O, (I), and N-(4-nitrophenyl)pyridine-2-carboxamide, C(12)H(9)N(3)O(3), (II), have been studied. Compound (I) exhibits unconventional aryl-carbonyl C-H...O and pyridine-fluorine C-H...F hydrogen bonding in two dimensions and well defined π-stacking involving pyridine rings in the third dimension. The conformation of (II) is more nearly planar than that of (I) and the intermolecular interactions comprise one-dimensional aryl-carbonyl C-H...O hydrogen bonds leading to a stepped or staircase-like progression of loosely π-stacked molecules. The close-packed layers of planar π-stacked molecules are related by inversion symmetry. Two alternating interplanar separations of 3.439 (1) and 3.476 (1) Šare observed in the crystal lattice and are consistent with a repetitive packing sequence, ABA'B'AB…, for the π-stacked inversion pairs of (II).