Computational modeling and target synthesis of monomethoxy-substituted o-diphenylisoxazoles with unexpectedly high antimitotic microtubule destabilizing activity.

The ability of monomethoxy-substituted o-diphenylisoxazoles 2a-d to interact with the colchicine site of tubulin was predicted using computational modeling, docking studies, and calculation of binding affinity. The respective molecules were synthesized in high yields by three steps reaction using easily available benzaldehydes, acetophenones, and arylnitromethanes as starting material. The calculated antitubulin effect was confirmed in vivo in a sea urchin embryo model. Compounds 2a and 2c showed high antimitotic microtubule destabilizing activity compared to that of CA4. Isoxazole 2a also exhibited significant cytotoxicity against human cancer cells in NCI60 screen. For the first time, isoxazole-linked CA4 derivatives 2a and 2c with only one methoxy substituent were identified as potent antimitotic microtubule destabilizing agents. These molecules could be considered as promising structures for further optimization.

Structure-based inhibitors targeting the alpha-helical domain of the Spiroplasma melliferum histone-like HU protein.

Here we report bisphenol derivatives of fluorene (BDFs) as a new type of chemical probes targeting a histone-like HU protein, a global regulator of bacterial nucleoids, via its dimerization interface perturbation. BDFs were identified by virtual screening and molecular docking that targeted the core of DNA-binding ß-saddle-like domain of the HU protein from Spiroplasma melliferum. However, NMR spectroscopy, complemented with molecular dynamics and site-directed mutagenesis, indicated that the actual site of the inhibitors' intervention consists of residues from the α-helical domain of one monomer and the side portion of the DNA-binding domain of another monomer. BDFs inhibited DNA-binding properties of HU proteins from mycoplasmas S. melliferum, Mycoplasma gallicepticum and Escherichia coli with half-maximum inhibitory concentrations in the range between 5 and 10 µM. In addition, BDFs demonstrated antimicrobial activity against mycoplasma species, but not against E. coli, which is consistent with the compensatory role of other nucleoid-associated proteins in the higher bacteria. Further evaluation of antimicrobial effects of BDFs against various bacteria and viruses will reveal their pharmacological potential, and the allosteric inhibition mode reported here, which avoids direct competition for the binding site with DNA, should be considered in the development of small molecule inhibitors of nucleoid-associated proteins as well as other types of DNA-binding multimeric proteins.

Novel curcumin derivatives as P-glycoprotein inhibitors: Molecular modeling, synthesis and sensitization of multidrug resistant cells to doxorubicin.

The MDR1/P-glycoprotein (Pgp)/ABCB1 multidrug transporter is being investigated as a druggable target for antitumor therapy for decades. The natural product curcumin is known to provide an efficient scaffold for compounds capable of blocking Pgp mediated efflux and sensitization of multidrug resistant (MDR) cells to the Pgp transported drug doxorubicin (Dox). We performed molecular dynamics simulations and docking of curcumin derivatives into the Pgp model. Based on these calculations, a series of pyrazolocurcumin derivatives with predicted metabolic stability and/or improved binding affinity were proposed for synthesis and evaluation of MDR reversal potency against Dox selected K562/4 subline, a derivative of K562 human chronic myelogenous leukemia cell line. Compounds 16 and 19 which are both dimethylcurcumin pyrazole derivatives bearing an N-p-phenylcarboxylic amide substitution, were the most potent Pgp blockers as determined by intracellular Dox accumulation. Furthermore, at non-toxic submicromolar concentrations 16 and 19 dramatically sensitized K562/4 cells to Dox. Together with good water solubility of 16 and 19, these results indicate that the new pyrazolo derivatives of curcumin are a promising scaffold for development of clinically applicable Pgp antagonists.

An explicit account of solvation is essential for modeling Suzuki-Miyaura coupling in protic solvents.

We compared explicit and implicit solvation approaches in modeling the free energy profile of the final step of Suzuki-Miyaura coupling. Both approaches produced similar ΔG(≠) in all the studied solvents (benzene, toluene, DMF, ethanol, and water). Solvation free energies of individual reaction components reasonably correlated for explicit and implicit models in aprotic solvents (RMSE = 30-50 kJ mol(-1), R(2) > 0.71). However for ethanol and water the correlation was poor. We attributed this difference to the formation of the PdH-O hydrogen bond with Pd(PPh3)2 which was surprisingly observed in explicit modeling. Further QM calculations of the Pd(PPh3)2-H2O system confirmed the direction (PdH) and stability of this bonding. Therefore we stress the need for considering explicit solvation for modeling Pd-catalyzed reactions in protic solvents.

2,3-Dihydroxy-quinoxaline induces ATPase activity of Herpes Simplex Virus thymidine kinase.

2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with Ki=250 µM (95% CI: 106-405 µM) and dose-dependently increased the rate of the ATP hydrolysis with KM=112 µM (95% CI: 28-195 µM). The kinetic scheme consistent with this experimental data is proposed.

Hit clustering can improve virtual fragment screening: CDK2 and PARP1 case studies.

Virtual fragment screening could be a promising alternative to existing experimental screening techniques. However, reliable methods of in silico fragment screening are yet to be established and validated. In order to develop such an approach we first checked how successful the existing molecular docking methods can be in predicting fragment binding affinities and poses. Using our Lead Finder docking software the RMSD of the binding energy prediction was observed to be 1.35 kcal/mol(-1) on a set of 26 experimentally characterized fragment inhibitors, and the RMSD of the predicted binding pose from the experimental one was <1.5 Å. Then, we explored docking of 68 fragments obtained from 39 drug molecules for which co-crystal structures were available from the PDB. It appeared that fragments that participate in oriented non-covalent interactions, such as hydrogen bonds and metal coordination, could be correctly docked in 70-80% of cases suggesting the potential success of rediscovering of corresponding drugs by in silico fragment approach. Based on these findings we've developed a virtual fragment screening technique which involved structural filtration of protein-ligand complexes for specific interactions and subsequent clustering in order to minimize the number of preferable starting fragment candidates. Application of this method led to 2 millimolar-scale fragment PARP1 inhibitors with a new scaffold.