In pharmaceutical industry, lead discovery strategies and screening collections have been predominantly tailored to discover compounds that modulate target proteins through noncovalent interactions. Conversely, covalent linkage formation is an important mechanism for a quantity of successful drugs in the market, which are discovered in most cases by hindsight instead of systematical design. In this article, the implementation of a docking-based virtual screening workflow for the retrieval of covalent binders is presented considering human cathepsin K as a test case. By use of the docking conditions that led to the best enrichment of known actives, 44 candidate compounds with unknown activity on cathepsin K were finally selected for experimental evaluation. The most potent inhibitor, 4-(N-phenylanilino)-6-pyrrolidin-1-yl-1,3,5-triazine-2-carbonitrile (CP243522), showed a K(i) of 21 nM and was confirmed to have a covalent reversible mechanism of inhibition. The presented approach will have great potential in cases where covalent inhibition is the desired drug discovery strategy.

ObjectiveTo identify a novel class of inhibitors of fungal transporters involved in drug resistance.MethodsA series of structurally-related low molecular mass compounds was synthesized using combinatorial chemistry of a cyclobutene-dione (squarile) core. These compounds were screened for their inhibition of plasma membrane Major Facilitator Superfamily (MFS) and ATP-binding cassette (ABC) transporters responsible for efflux pump-mediated drug resistance in the fungal pathogen Candida albicans. Strains of Saccharomyces cerevisiae that specifically overexpress the MFS pump CaMdr1p or the ABC transporter CaCdr1p were used in primary screens and counterscreens, respectively, and to detect inhibition of glucose-dependent Nile Red efflux. Efflux pump inhibition, activity as pump substrates and antifungal activity against yeast and clinical isolates expressing efflux pumps were determined using agarose diffusion susceptibility assays and checkerboard liquid chemosensitization assays with fluconazole.ResultsThe screen identified five structurally-related compounds which inhibited CaMdr1p. Two compounds, A and B, specifically chemosensitized AD/CaMDR1 to FLC in a pH-dependent fashion and acted synergistically with FLC in checkerboard liquid MIC assays but compound B had limited solubility. Compound A chemosensitized to FLC the azole-resistant C. albicans strain FR2, which over-expresses CaMdr1p, inhibited Nile Red efflux mediated by CaMdr1p but not CaCdr1p and was not toxic to cultured human cells. A minor growth-inhibitory effect of B on AD/CaMDR1, but not on AD/CaCDR1 and AD/CaCDR2, indicated that compound B may be a substrate of these transporters. The related compound F was found to have antifungal activity against the three pump over-expressing strains used in the study.ConclusionsCompound A is a ‘first in class’ small molecule inhibitor of MFS efflux pump CaMdr1p.

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Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis. This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest—a phenomenon known as mitotic catastrophe, which is followed by immediate cell death via apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in vivo in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.

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In the facultatively anaerobic bacterium Bacillus licheniformis a gene encoding a protein of the fumarate nitrate reductase family of transcriptional regulators (Fnr) was isolated. Unlike Fnr proteins from gram-negative bacteria, but like Fnr fromBacillus subtilis, the protein contained a C-terminal cluster of cysteine residues. Unlike in Fnr from B. subtilis, this cluster (Cys226-X2-Cys229-X4-Cys234) is composed of only three Cys residues, which are supposed to serve together with an internal residue (Cys71) as the ligands for an FeS center. Transfer of the B. licheniformis gene to anfnr mutant of B. subtilis complemented the ability for synthesis of nitrate reductase during anaerobic growth.

Multidrug resistance (MDR) in tumors and pathogens remains a major problem in the efficacious treatment of patients by reduction of therapy options and subsequent treatment failure. Various mechanisms are described to be involved in the development of MDR with overexpression of ATP-binding cassette (ABC) transporters reflecting the most extensively studied. These membrane transporters translocate a wide variety of substrates utilizing energy from ATP hydrolysis leading to decreased intracellular drug accumulation and impaired drug efficacy. One treatment strategy might be inhibition of transporter-mediated efflux by small molecules. Isocoumarins and 3,4-dihydroisocoumarins are a large group of natural products derived from various sources with great structural and functional variety, but have so far not been in the focus as potential MDR reversing agents. Thus, three natural products and nine novel 3,4-dihydroisocoumarins were designed and analyzed regarding cytotoxicity induction and inhibition of human ABC transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) in a variety of human cancer cell lines as well as the yeast ABC transporter Pdr5 in Saccharomyces cerevisiae . Dual inhibitors of P-gp and BCRP and inhibitors of Pdr5 were identified, and distinct structure-activity relationships for transporter inhibition were revealed. The strongest inhibitor of P-gp and BCRP, which inhibited the transporters up to 80 to 90% compared to the respective positive controls, demonstrated the ability to reverse chemotherapy resistance in resistant cancer cell lines up to 5.6-fold. In the case of Pdr5, inhibitors were identified that prevented substrate transport and/or ATPase activity with IC 50 values in the low micromolar range. However, cell toxicity was not observed. Molecular docking of the test compounds to P-gp revealed that differences in inhibition capacity were based on different binding affinities to the transporter. Thus, these small molecules provide novel lead structures for further optimization.

Nature is an indispensable source of new drugs, providing unique bioactive lead structures for drug discovery. In the present study, secalonic acid F (SAF), a naturally occurring ergochrome pigment, was studied for its cytotoxicity against various leukemia and multiple myeloma cells by the resazurin assay. SAF exhibited cytotoxic activity on both leukemia and multiple myeloma cells. Generally, multiple myeloma cells were more sensitive to SAF than leukemia cells. NCI-H929 cells were the most affected cells among the tested panel of multiple myeloma cell lines and were taken for further studies to assess the mode of action of SAF on those cells. Cell cycle analysis revealed that SAF induced S and G2/M arrest in NCI-H929 cells. SAF-associated apoptosis and necrosis resulted in cytotoxicity. SAF further inclined the disassembly of the tubulin network, which may also account for its cytotoxicity. COMPARE and hierarchical cluster analyses of transcriptome-wide expression profiles of the NCI tumor cell line panel identified genes involved in numerous cellular processes (e.g., cell differentiation, cell migration, and other numerous signaling pathways) notably correlated with log10IC50 values for secalonic acid. In conclusion, the present study supports the therapeutic potential of SAF to treat multiple myeloma.