Combinatorial Chemistry & High Throughput Screening - Volume 17, Issue 7, 2014

It is well accepted and hopefully understood that a global crisis is looming on the horizon to do mainly with the decline and limitation of our current drug arsenal to fight existing and newly diagnosed diseases. An example would be the ongoing Ebola epidemic in West Africa leaving doctors unsure on how to stop this deadliest outbreak; and governments, especially in Europe and America, even more concerned about the virus and its likely possibility of making it to their shores - no arsenal is available today, unfortunately. Another one is the newly discovered single-stranded RNA species of the genus Betacoronavirus, commonly known as the Middle East respiratory syndrome coronavirus (MERS-CoV); this virus has already travelled as far away as the US from the kingdom of Saudi Arabia - again no arsenal to stop it. Without strong commitment for basic and applied research from worldwide leaders and their governments combined with policed research ethics eliminating issues surrounding biomedical research data reproducibility and lack of transparency, vulnerabilities will become our worst enemy. The mere call for an "international response to a drastic situation" by the World Health Organization to contain the spread of Ebola is a testament. Discovering drugs to fight disease is a complicated web of arrows to follow, boxes to tick, market size assessment, intellectual property ownership, and regulatory hurdles to jump; often with poor outcomes in the form of failures in first-in-man clinical trials; Pharma companies are by far the experts in this business of drug discovery and development; yet they keep failing to contribute to the growth of our global drug arsenal to fight disease - instead they seem to spend their hard earned cash on acquiring other companies, pleasing analysts, investors, and shareholders alike by "reducing expenditure and cost to do business". In reality, they are killing true innovation at the workplace and introducing a sense of job insecurity within the various ranks at their companies, in my opinion, positioning themselves on a dark course towards extinction. The recent emergence of drug discovery efforts within academic institutions, driven primarily by the road map initiative of the National Institute of Health (USA), have been a fresh sign of change and empowerment in the right direction - allowing academics to tinker with biology using chemistry for their research and if discoveries pertinent to drugs were to be made, then one could imagine the private sector stepping in to further develop them into products under the framework of "private-public" collaborations. It is still too earlier to make a judgment call as to whether these initiatives were successful or not; as this would provide a proven and validated business model to build our arsenal, except perhaps to caution against the raise of "greed" within these institutions which could inherently hinder partnerships. Beauty is always in the eye of the beholder; more so in academia and often making it difficult to form partnerships as due diligence on their research is always unpleasant to hear critiques and reality checks, especially with relation to the worthiness of their work; way different from the classical process of publishing in high profile journals - only for many to struggle with reproducing their published findings. I am cautiously optimistic that funding chemical biology and allowing researchers to publish, would provide an avenue to assess the size and geographical locations of the global efforts enabling both future intercontinental collaborations and rapid international response to outbreaks. We, at Combinatorial Chemistry and High Throughput Screening, are making every effort to support such global initiatives through publishing diverse research from worldwide research group under the framework of good science as eluded to by our Editor-in-Chief, Dr. Rathnam Chaguturu in his annual editorial earlier in the year. This issue contains seven distinct research articles covering organic synthetic chemistry to make novel molecules to fight microbes and viruses, virtual screening for inhibitors of Yersinia Outer Protein H to combat this gram negative bacillus, tackling the challenges of searching for small molecule inhibitors of the RNA binding protein MUSHASI to fight cancer, assessing perfume raw materials for their antimicrobial activity, and one concise review on the use of label free technologies for phenotypic drug discovery screening. In all, this issue contains research efforts from six countries and three continents: India, Iran, Poland, Serbia, Switzerland, and USA.

Phenotypic screen holds great potential in the discovery of new small molecule probes and drugs, as it permits interrogating small molecules with native targets and pathways in model organisms and disease relevant cells. In recent years, label-free cell phenotypic profiling has emerged as an alternative for drug discovery. This paper provides the rationale for phenotypic screens, discusses the basics of label-free cell phenotypic profiling technologies, and provides some guidelines as to how to use these techniques to facilitate drug discovery.

Yersinia pestis, a Gram negative bacillus, spreads via lymphatic to lymph nodes and to all organs through the bloodstream, causing plague. Yersinia outer protein H (YopH) is one of the important effector proteins, which paralyzes lymphocytes and macrophages by dephosphorylating critical tyrosine kinases and signal transduction molecules. The purpose of the study is to generate a three-dimensional (3D) pharmacophore model by using diverse sets of YopH inhibitors, which would be useful for designing of potential antitoxin. In this study, we have selected 60 biologically active inhibitors of YopH to perform Ligand based pharmacophore study to elucidate the important structural features responsible for biological activity. Pharmacophore model demonstrated the importance of two acceptors, one hydrophobic and two aromatic features toward the biological activity. Based on these features, different databases were screened to identify novel compounds and these ligands were subjected for docking, ADME properties and Binding energy prediction. Post docking validation was performed using molecular dynamics simulation for selected ligands to calculate the Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF). The ligands, ASN03270114, Mol_252138, Mol_31073 and ZINC04237078 may act as inhibitors against YopH of Y. pestis.

RNA-binding proteins (RBPs) can act as stem cell modulators and oncogenic drivers, but have been largely ignored by the pharmaceutical industry as potential therapeutic targets for cancer. The MUSASHI (MSI) family has recently been demonstrated to be an attractive clinical target in the most aggressive cancers. Therefore, the discovery and development of small molecule inhibitors could provide a novel therapeutic strategy. In order to find novel compounds with MSI RNA binding inhibitory activity, we have developed a fluorescence polarization (FP) assay and optimized it for high throughput screening (HTS) in a 1536-well microtiter plate format. Using a chemical library of 6,208 compounds, we performed pilot screens, against both MSI1 and MSI2, leading to the identification of 7 molecules for MSI1, 15 for MSI2 and 5 that inhibited both. A secondary FP dose-response screen validated 3 MSI inhibitors with IC50 below 10 μM. Out of the 25 compounds retested in the secondary screen only 8 demonstrated optical interference due to high fluorescence. Utilizing a SYBR-based RNA electrophoresis mobility shift assay (EMSA), we further verified MSI inhibition of the top 3 compounds. Surprisingly, even though several aminoglycosides were present in the library, they failed to demonstrate MSI inhibitor activity challenging the concept that these compounds are pan-active against RBPs. In summary, we have developed an in vitro strategy to identify MSI specific inhibitors using an FP HTS platform, which will facilitate novel drug discovery for this class of RBPs.

An efficient, one-pot and three-component synthesis of biologically important heterocyclic compounds is described from the reaction of primary amines and phenyl isothiocyanate in the presence of acryloyl chloride at room temperature without the need to use any catalyst.

A microdilution protocol was developed and automated using a liquid handling station, allowing the determination of minimum inhibitory concentrations (MIC) of hydrophobic raw materials commonly used in the perfume industry (essential oils and synthetic chemicals). Tests were performed in 96-well microtiter plates against standard bacterial test strains and skin isolates involved in underarm malodor. The comparison with data previously reported in the literature indicated that the protocol was suitable, yielding MIC values that were in general agreement with those derived from manual test methods. For the majority of active test compounds, results showed a pronounced difference in susceptibility pattern between the Gram-positive and Gram-negative test strains used in this study. For a group of acyclic aliphatic aldehydes, a structureactivity relationship depending on the chain length was found.

Application of chemometric methods in the study of the retention indices of thirteen 5-arylidene-2,4- thiazolidinediones in two low polarity high-performance thin-layer chromatographic (HPTLC) stationary phases (RP-18 and RP-CN) and six aqueous mobile phases. Principal component analysis classified chromatographic systems into four specific groups while one system remained non-classified. Hierarchical clustering analysis enabled grouping of the chromatographic systems into three clusters, and the studied compounds into three main classes. The color map enabled more in-depth interpretation of the relationships between the studied compounds and HPTLC systems applied.

A series of structurally novel, (E)-N'-(4-aryloxybenzylidene)-1H-benzimidazole-2-carbohydrazide derivatives were synthesized by molecular hybridization technique. All these compounds were evaluated against Mycobacterium tuberculosis H37Rv strains using Resazurin Microtiter assay (REMA) method. These compounds showed good antituberculosis activity with minimum inhibitory concentration (MIC) value of the range of 1.5-25 µg/mL.

Knowledge of the lipophilicity of candidate compounds for prodrugs may predict their predetermined course/effect in the body. Acyclovir (ACV) belongs to a class of drugs with low bioavailability. Its tricyclic analogues, the derivatives of 3,9-dihydro-3-[(2-hydroxyethoxy)methyl]-9-oxo-5H-imidazo[1,2-a]purine (TACV) exhibit similar antiviral activities and are more lipophilic as compared with acyclovir itself. In the search for new antiviral prodrugs 6-(4- methoxyphenyl) tricyclic compound (6-(4-MeOPh)-TACV) was modified by esterification of a hydroxyl group in the aliphatic chain. Selected esters (acetyl, isobutyryl, pivaloyl, ethoxycarbonyl and nicotinoyl) were synthesized and their lipophilicity was determined by the HPLC-RP method. The study compared the log kw calculated from the linear and quadratic equations and proved the correctness of the application of the linear relationship log k as a function of the concentration of ACN in the mobile phase (30-60%). Statistical analyses of the comparative values of log kw and clogP were carried out using computational methods. It was proved that the AC logP algorithm can be useful for the analysis of these compounds, which can have a statistically justified application in the assessment of the quantitative structure– activity relationship (QSAR). The lipophilicity determined by the HPLC method appears as follows: 6-(4-MeOPh)-TACV < Ac- < Nic- < Etc- < iBut- < Piv- (log kw = 0.65-2.26). Finally, the HPLC-RP method was developed and validated for simultaneous determination of synthesized esters.