Current Drug Discovery Technologies (v.13, #1)

Meet Our Editorial Board Member by Melgardt M. De Villiers (1-1).

Protein kinases are an important class of enzymes and drug targets. New opportunities to discover medicines for neglected diseases can be leveraged by the extensive kinase tools and knowledge created in targeting human kinases. A valuable tool for kinase drug discovery is an enzyme assay that measures catalytic function. The functional assay can be used to identify inhibitors, estimate affinity, characterize molecular mechanisms of action (MMOAs) and evaluate selectivity. However, establishing an enzyme assay for a new kinases requires identification of a suitable substrate. Identification of a new kinase's endogenous physiologic substrate and function can be extremely costly and time consuming. Fortunately, most kinases are promiscuous and will catalyze the phosphotransfer from ATP to alternative substrates with differing degrees of catalytic efficiency. In this manuscript we review strategies and successes in the identification of alternative substrates for kinases from organisms responsible for many of the neglected tropical diseases (NTDs) towards the goal of informing strategies to identify substrates for new kinases. Approaches for establishing a functional kinase assay include measuring auto-activation and use of generic substrates and peptides. The most commonly used generic substrates are casein, myelin basic protein, and histone. Sequence homology modeling can provide insights into the potential substrates and the requirement for activation. Empirical approaches that can identify substrates include screening of lysates (which may also help identify native substrates) and use of peptide arrays. All of these approaches have been used with a varying degree of success to identify alternative substrates.

Antigens of infectious agents share amino acid sequences with human proteins. Such a peptide matching may lead to autoimmunity through crossreactivity phenomena following pathogen infection and/or immunotherapeutic approaches. In this framework, we analyzed as a model the primary sequence of human cytomegalovirus (HCMV) glycoprotein B (gB) protein and searched for viral peptide sequences shared with human proteins. We show that the HCMV antigen has a high peptide identity with a large number of human proteins at the penta-, hexa-, and heptapeptide level, with the viral versus human peptide overlap involving host proteins implicated in crucial processes such as embryonic development, spermatogenesis, spatial learning, and hippocampal plasticity, inter alia. This study might help understand the etiology of the pathologic sequela associated with HCMV (re)activation and, in addition, address scientific and clinical research toward the definition of antiviral therapeutics based on non-crossreactive viral sequences.

Anti-Aging Property of G2013 Molecule as a Novel Immunosuppressive Agent on Enzymatic and Non-Enzymatic Oxidative Stress Determinants in Rat Model by Abbas Mirshafiey, Soma Hosseini, Sanaz Afraei, Noshin Rastkari, Farzaneh T. Zavareh, Gholamreza Azizi (25-33).
Background: G2013 molecule is a novel non-steroidal anti-inflammatory agent with immunosuppressive property, which was investigated on determinants relative to the oxidative stress in animal model. Materials and Methods: The Sprague-Dawley rats were used for evaluating properties of G2013 on some oxidative stress enzymes including: Myeloperoxidase (MPO), Glutathione peroxidase (GPX1), mitochondrial Superoxide dismutase (SOD2), Catalase (CAT), Glutathione S-Transferase (GST), and inducible nitric oxide synthase (iNOS) genes expression by Real Time PCR. The rats were sacrificed 3 months after daily oral administration of G2013. Moreover, Malondialdehyde (MDA), Carbonyl protein (PCO), the lipid and protein oxidation markers respectively and total anti-oxidant capacity (TAC) were tested in serum by biochemical analysis. Also cortisol as a steroid hormone was evaluated by chemiluminescence immunoassay after 12 weeks consumption of G2013 solution. Result: Our findings revealed a significant decrease in MPO in G2013 treated group, indicating its favorable effects but has no significant effects on genes expression of another antioxidant enzymes, including: SOD2, CAT, GPX1, and GST. Also, there were no significant differences in PCO, TAC and cortisol compared to control group following G2013 consumption. While an enhancement in serum MDA level was observed in the treatment group. In addition, G2013 therapy did not show any weight loss. Conclusions: Our data showed the safety and efficacy of G2013 as a novel designed NSAID on various oxidative stress determinants.

Relative In Vitro Potentials of Parthenolide to Induce Apoptosis and Cell Cycle Arrest in Skin Cancer Cells by Vazhappilly C. George, Devanga R.N. Kumar, Rangasamy A. Kumar (34-40).
Background: Parthenolide (PN) has been reported to inhibit proliferation and induces apoptosis in a variety of cancer cells with different mechanisms. Nevertheless, to the best of our knowledge, its relative anticancer activity at lower doses has not been reported in HaCaT immortalized keratinocytes and A375 melanoma cells.
Objective and Method: The present study aimed to investigate the relative anticancer effects of PN by analysing its cytotoxicity, apoptosis and cell cycle arresting potentials in HaCaT and A375 cells.
Result: PN was found to be significantly cytotoxic with lower IC50 values of 1.45 µM and 2.9 µM (p<0.05) in HaCaT and A375 cells, respectively with nuclear disruption as evident by DAPI staining. A dose-dependent increase in DNA fragmentation indicated apoptotic cell death. Furthermore, the percentage of cells deposition at S (35.56 to 43.68%) and G0-G1 (27.15 to 56.44%) phases after 24 h of PN treatment, substantiate its cell cycle arresting potentials in HaCaT and A375 cells.
Conclusion: These comparative results provided substantial evidence to conclude the anticancer potential of PN, especially at lower dosages to induce cell death mechanisms in skin epidermal cells.

Ester Prodrugs of Ketoprofen: Synthesis, In Vitro Stability, In Vivo Biological Evaluation and In Silico Comparative Docking Studies Against COX-1 and COX-2 by Musa Ahmed, Faizul Azam, Abdul Gbaj, Abdulmottaleb E. Zetrini, Amna S. Abodlal, Abir Rghigh, Eman Elmahdi, Amel Hamza, Mabruk Salama, Salah M. Bensaber (41-57).
Prompted by the ineptness of the currently used non-steroidal antiinflammatory drugs (NSAIDs) to control gastric mucosal and renal adverse reactions, several ester prodrugs of ketoprofen were synthesized and characterized by IR, 1H NMR and mass spectral data. Physicochemical properties such as aqueous solubility, octanol-water partition coefficient log P, chemical stability and enzymatic hydrolysis of the synthesized molecules have been studied to assess their potential as prodrugs. The obtained results confirmed that all ester prodrugs are chemically stable, possess increased lipophilicity compared to their parent compounds and converted to the active drugs in vivo. All of the tested ester prodrugs exhibited marked anti-inflammatory activity ranging from 91.8% to 113.3% in comparison with the parent drug, ketoprofen. A mutual prodrug obtained from two antiinflammatory molecules, ketoprofen and salicylic acid has been noted to potentiate the activity making it most active molecule of the series. The ulcerogenic index of the ester prodrugs was significantly lower than the parent drug, ketoprofen. Comparative docking studies against X-ray crystal structures of COX-1 and COX-2 further provided understanding of their interaction with the cyclooxygenases that will facilitate design of better inhibitors (or prodrugs) with sufficient specificity for COX-2 against COX-1. The study offers an innovative strategy for finding a molecule with safer therapeutic profile for longterm treatment of inflammatory diseases.