A combination strategy of structure-based virtual screening, MM-GBSA, cross docking, molecular dynamics and metadynamics simulations used to investigate natural compounds as potent and specific inhibitors of tumor linked human carbonic anhydrase IX.

Cancer remains a serious health concern representing one of the leading causes of deaths worldwide. The enzyme human carbonic anhydrase IX (hCA IX) is found to be over-expressed in many cancer types and its selective inhibition over its cytosolic off-target isoform, human carbonic anhydrase II (hCA II), represents a potential area of research in the development of novel anticancer compounds. This work is concerned with the use of various in silico tools for the identification of natural product based molecules that can selectively inhibit hCA IX over hCA II. MM-GBSA assisted structure-based virtual screening against hCA IX was performed for nearly 225,000 natural products imported from the ZINC15 database. The obtained hits were checked for their potency by considering acetazolamide, the bound inhibitor of hCA IX, as the reference molecule, and 121 molecules were identified as potent hCA IX inhibitors. After ensuring their potency, cross-docking, followed by MM-GBSA calculations of the hits with hCA II, was performed, and their selectivity was assessed by considering the hCA IX selective compound SLC-0111 as the reference molecule, and 50 natural products were identified as potent as well as selective hCA IX inhibitors. Molecules with the quinoline scaffold showed the highest selectivity, and their selectivity was attributed to the strong electrostatic interactions of the zinc binding group (ZBG) with the active site Zn(II) ion. Furthermore, the stability of the binding modes of the top hCA IX selective hits was ensured by performing molecular dynamics (MD) simulations, which clearly proved that one of the short-listed molecules is truly selective, as it does not interact with the active site Zn(II) ion of hCA II, but interacts strongly with this ion in hCA IX. Bonding pose metadynamics studies revealed that the ligand moves to a more stable binding site from the one predicted by the docking studies and shows stronger interaction with the protein and Zn(II) at this binding site. The ligand is not likely to have issues with bioavailability. As a result, this ligand can be taken for bioassay testing and subsequently used as a feasible therapeutic treatment for a variety of cancer types. Communicated by Ramaswamy H. Sarma.

Identification of potent human carbonic anhydrase IX inhibitors: a combination of pharmacophore modeling, 3D-QSAR, virtual screening and molecular dynamics simulations.

Human carbonic anhydrase IX (hCA IX) is a promising target for the development of potential anticancer agents. In the current study, pharmacophore and 3D-QSAR models have been developed using SLC-0111 derivatives. The developed models have been further utilized for the virtual screening process to develop potent hCA IX inhibitors. Thirteen different models have been developed by employing various combinations of training and test set molecules. Based on this, a model, AADDR.135, comprising two H-bond acceptors, two H-bond donors and one aromatic ring, has been found as the best QSAR model. The proposed model exhibits high robustness (R2 = 0.9789), with good predictive ability (Q2 = 0.6872). An external library of drug-like compounds (∼10000 molecules) imported from the ZINC15 database has been screened over the model AADDR.135. In total, 1601 compounds were obtained as hits. Molecular docking studies and molecular dynamics simulations have been performed on the obtained hits and, based on these computations, two unique molecules have been identified as potential hCA IX inhibitors. These show higher binding energies compared to the parent molecule and its most potent analogue.Communicated by Ramaswamy H. Sarma.

A comparative study of different docking methodologies to assess the protein-ligand interaction for the E. coli MurB enzyme.

We have investigated the active site of E. coli MurB using the Quantum Mechanics/Molecular Mechanics (QM/MM) methodology. The docking of three novel series of 4-thiazolidinone derivatives has been performed using two methods: rigid docking and flexible docking (Induced Fit Docking: IFD). The results have been compared to understand the conformational aspects of the enzyme. The docking results from rigid docking show that the ligands with highly negative ΔGbind have poor docking scores. In addition, the value of the regression coefficient (R) obtained on correlating the ΔGbind and the experimental pMIC values is insignificant. On keeping the protein flexible, there is a remarkable improvement in both the docking score and ΔGbind, along with a good value of R (0.64). Two important residues, Tyr254 and Try190 are found to be highly displaced during the flexible docking and hence their role in effective ligand binding has been confirmed. Thus, comparing the two methodologies, IFD has emerged as the more appropriate one for studying the E. coli MurB enzyme. To further substantiate the findings, MD studies over a time period of 20 ns have been performed on the IFD-LIII j and Rigid/XP-LIII j complexes and the results shows the former complex to be more stable, with lower average RMSD and higher average ΔGbind.Communicated by Ramaswamy H. Sarma.

Structure-based virtual screening, free energy of binding and molecular dynamics simulations to propose novel inhibitors of Mtb-MurB oxidoreductase enzyme.

Currently, the growing incidence of drug resistance toward tuberculosis intensified the need for discovery of novel targets and their inhibitors. The enzyme MurB which is involved in one of the steps for peptidoglycan biosynthesis is an effective target that can produce drugs having lesser side-effects. Recently the only crystal structure of Mycobacterium Tuberculosis MurB has been deposited and, therefore, in the present study, we have used this as a target for virtual screening of drug-like molecules from the ZINC Database. We have also designed a complete workflow for the process which resulted in 12 hit compounds that have good docking scores, ΔGbind, and Glide energy. The hits obtained have also been found to share structural features with some known antibiotics such as Amoxicillin. Furthermore, MD simulations on the top most hit L1 displayed its stable binding with the enzyme. Thus, this study has proved helpful in proposing novel inhibitors for MurB enzyme that can be tested against various TB strains.

A comparative study of the binding modes of SLC-0111 and its analogues in the hCA II and hCA IX active sites using QM/MM, molecular docking, MM-GBSA and MD approaches.

Human carbonic anhydrase IX (hCA IX) is over-expressed in many tumor types and serves as an important target for the discovery of novel anticancer agents. However, development of compounds that can selectively inhibit hCA IX over its widespread cytosolic isoform human carbonic anhydrase II (hCA II) is a major challenge. This work focuses on recognizing the structural features of the hCA IX receptor that could help in achieving its selective inhibition. Tools such as protein structure alignment, rigid as well as flexible docking, QM/MM calculations and molecular dynamics simulations on SLC-0111, a selective hCA IX inhibitor, in complexation with each receptor, have been used to differentiate the receptor-ligand interactions in the two complexes. It is found that the ligand shows better binding to hCA IX due to stronger coordination to the Zn (II) ion. The ligand provides bidentate coordination through its negatively charged nitrogen and an oxygen of the sulfonamide zinc binding group. Binding energy calculations show that the potency of this ligand is due to the hydrophobic contacts, whereas the selectivity is due to the electrostatic interactions. Molecular docking and binding energy calculations for three different series of SLC-0111 analogs have identified a few molecules that show high potency and selectivity toward hCA IX. It is found that both hydrophobic and polar contacts contribute to the potency and selectivity of the ligands.

Highly Multiplexed Fluorescence in Situ Hybridization for in Situ Genomics.

The quantification of changes in gene copy number is critical to our understanding of tumor biology and for the clinical management of cancer patients. DNA fluorescence in situ hybridization is the gold standard method to detect copy number alterations, but it is limited by the number of genes one can quantify simultaneously. To increase the throughput of this informative technique, a fluorescent bar-code system for the unique labeling of dozens of genes and an automated image analysis algorithm that enabled their simultaneous hybridization for the quantification of gene copy numbers were devised. We demonstrate the reliability of this multiplex approach on normal human lymphocytes, metaphase spreads of transformed cell lines, and cultured circulating tumor cells. It also opens the door to the development of gene panels for more comprehensive analysis of copy number changes in tissue, including the study of heterogeneity and of high-throughput clinical assays that could provide rapid quantification of gene copy numbers in samples with limited cellularity, such as circulating tumor cells.