Discovery of new selective human aldose reductase inhibitors through virtual screening multiple binding pocket conformations

Aldose reductase reduces glucose to sorbitol. It plays a key role in many of the complications arising from diabetes. Thus, aldose reductase inhibitors (ARI) have been identified as promising therapeutic agents for treating such complications of diabetes, as neuropathy, nephropathy, retinopathy, and cataracts. In this paper, a virtual screening protocol applied to a library of compounds in house has been utilized to discover novel ARIs. IC50's were determined for 15 hits that inhibited ALR2 to greater than 50% at 50 uM, and ten of these have an IC50 of 10 uM or less, corresponding to a rather substantial hit rate of 14% at this level. The specificity of these compounds relative to their cross-reactivity with human ALR1 was also assessed by inhibition assays. This resulted in identification of novel inhibitors with IC50's comparable to the commercially available drug, epalrestat, and greater than an order of magnitude better selectivity

Molecular modeling studies on HIV-1 Reverse Transcriptase (RT) and Heat shock protein (Hsp) 90 as a potential anti-HIV-1 target.

Masters Degree. University of KwaZulu-Natal, Durban.Human immunodeficiency virus (HIV) infection is the leading cause of death globally. This dissertation addresses two HIV-1 target proteins namely, HIV-1 Reverse Transcriptase (RT) and Heat shock protein (Hsp) 90. More specifically for HIV-1 RT, a case study for the identification of potential inhibitors as anti-HIV agents was carried out. A more refined virtual screening (VS) approach was implemented, which was an improvement on work previously published by our group- “target-bound pharmacophore modeling approach”. This study generated a pharmacophore library based only on highly contributing amino acid residues (HCAAR), instead of arbitrary pharmacophores, most commonly used in the conventional approaches in literature. HCAAR were distinguished based on free binding energy (FBE) contributions, obtained using calculation from molecular dynamics (MD) simulations. Previous approaches have relied on the docking score (DS) to generate energy-based pharmacophore models. However, DS are reportedly unreliable. Thus we present a model for a per-residue energy decomposition (PRED), constructed from MD simulation ensembles generating a more trustworthy pharmacophore model which can be applied in drug discovery workflow. This approach was employed in screening for potential HIV-1 RT inhibitors using the pharmacophoric features of the compound GSK952. The complex was subjected to docking and thereafter MD simulations confirmed the stability of the system. Experimentally determined inhibitors with known HIV-RT inhibitory activity were used to validate the proposed protocol. Two potential hits ZINC46849657 and ZINC54359621 showed a significant potential with regards to FBE. Reported results obtained from this work confirm that this new approach is favourable to the future of drug design process. Hsp90 was recently discovered to play a vital role in HIV-1 replication. Thus has emerged, as a promising target for anti-HIV-1 drugs. The molecular mechanism of Hsp90 is poorly understood, thus the second study was aimed to address this issue and provide a clear insight to the inhibition mechanism of Hsp90. Reasonable continuous MD simulations were employed for both unbound and bound Hsp90 conformations, to understand the dimerization and inhibition mechanisms. Results demonstrated that coumermycin A1 (C-A1), a newly discovered Hsp90 inhibitor, binds at the CTD dimer of Hsp90 and lead to a significant separation between orthogonally opposed residues, such as Arg591.B, Lys594.A, Ser663.A, Thr653.B, Ala665.A, Thr649.B, Leu646.B and Asn669A. A Large difference in magnitudes was observed in the radius of gyration (Rg), per-residue fluctuation, root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) confirming a completely more flexible state for the unbound conformation associated with dimerization. Whereas, a less globally correlated motion in the case of the bound conformer of Hsp90 approved a reduction of the dimeric process. This undoubtedly underlines the inhibition process due to ligand binding. The detailed dynamic analyses of Hsp90 presented herein are believed to give a greater insight and understanding to the function and mechanisms of inhibition of Hsp90. The report on the inhibitor-binding mode would also be of great assistance in the design of prospective inhibitors against Hsp90 as potential HIV target

Discovery of New Selective Human Aldose Reductase Inhibitors through Virtual Screening Multiple Binding Pocket Conformations

Aldose reductase reduces glucose to sorbitol. It plays a key role in many of the complications arising from diabetes. Thus, aldose reductase inhibitors (ARI) have been identified as promising therapeutic agents for treating such complications of diabetes, as neuropathy, nephropathy, retinopathy, and cataracts. In this paper, a virtual screening protocol applied to a library of compounds in house has been utilized to discover novel ARIs. IC₅₀’s were determined for 15 hits that inhibited ALR2 to greater than 50% at 50 μM, and ten of these have an IC₅₀ of 10 μM or less, corresponding to a rather substantial hit rate of 14% at this level. The specificity of these compounds relative to their cross-reactivity with human ALR1 was also assessed by inhibition assays. This resulted in identification of novel inhibitors with IC₅₀’s comparable to the commercially available drug, epalrestat, and greater than an order of magnitude better selectivity

Synthesis of aspartic proteases probes and their application for interaction identification and binding hotspots mapping

Aspartic proteases play a crucial role in human physiology and pathologyincluding as biomarkers for breast cancer and Alzheimer's disease, and as potential drug targets for infectious diseases. However, chemical probes for photoaffinity labeling (PAL) of these proteases are underdeveloped. We develop a full on-resin synthesis of clickable PAL probes based on the natural product inhibitor pepstatin, incorporating a minimal diazirine photo-reactive group. The positioning of this group in the inhibitor determines the labeling efficiency. Effective probes sensitively detect cathepsin D, a biomarker for breast cancer, in cell lysates. Through chemical proteomics experiments and deep learning algorithms, we also identify sequestosome-1 as a direct interaction partner and substrate of cathepsin D. PAL combined with tandem mass spectrometry (MSn) can reveal interactions between small molecule drugs and protein in biological environments. However, the direct detection of the ‘hotspots’ of the photo-crosslinking sites by MSn is challenging because of the unexpected fragmentation of small molecule drugs, especially when these are small peptides. We synthesize and introduce sulfoxide diazirine (SODA) building blocks to peptide-like probes for PAL. Those MS-cleavable probes enable a MS2 cleavage event that generates a probe-derived reporter ion and a minimal fragment on the modified peptide. Following a subsequent MS3 fragmentation event, we show that this strategy allows for unbiased identification of modification sites and mapping of binding hotspots of peptide-like bio-active molecules. Overall, our study presents the synthesis of aspartic proteases probes and their application for interaction identification and binding hotspots mapping. However, further improvement is required for this study to achieve broader application. We propose a number of possible follow-up experiments and discuss future prospects in chapter 6.Aspartatproteasen spielen eine entscheidende Rolle in der menschlichen Physiologie und Pathologie, unter anderem als Biomarker für Brustkrebs und Alzheimer, sowie als potenzielle Wirkstoffziele für Infektionskrankheiten. Allerdings ist die Toolbox chemischer Sonden für die Photoaffinitätsmarkierung (PAL) dieser Proteasen nur unzureichend entwickelt. Wir entwickeln eine vollständige On-Resin-Synthese für klickbare PAL-Sonden auf der Basis des Naturstoff-Inhibitors Pepstatin, die eine minimale Diazirin-Reaktivgruppe tragen. Es wurde gezeigt, dass die Platzierung der Gruppe im Inhibitor die Markierungseffizienz bestimmt und, dass diese Sonden den sensitiven Nachweis von Cathepsin D, einem Biomarker für Brustkrebs, in Zelllysaten erlauben. Dafür wurde eine Serie von Experimenten in Verbindung mit Deep-Learning-Algorithmen-gestützter Datenauswertung durchhgeführt, wobei Sequestrosom-1 als direkter Interaktionspartner und Substrat von Cathepsin D identifiziert wurde. Somit wurde gezeigt, dass PAL in Kombination mit Tandem-Massenspektrometrie (MSn) eingesetzt werden kann, um Interaktionen zwischen kleinen Moleküldrogen und Proteinen in biologischen Umgebungen aufzudecken. Die direkte Detektion der "Hotspots" der Photo-Crosslink-Stellen durch MSn ist jedoch aufgrund der komplexen und nicht vorhersehbaren Fragmentierung von kleinen Moleküldrogen, insbesondere bei kleinen Peptiden, weiterhin eine Herausforderung. Wir synthetisieren und führen Sulfoxid-Diazirin (SODA) Bausteine in Peptid-ähnliche Sonden für PAL ein. Dabei entsteht eine MS-labile Gruppe, die im MS2 zu einem ein Reporterion und ein modifiziertes Peptidion führt. Durch eine anschließende MS3 Fragmentierung zeigen wir, dass diese Strategie eine Identifizierung von Modifikationsstellen und die Kartierung von Bindungshotspots von Peptid-ähnlichen bioaktiven Molekülen ermöglicht. Zusammenfassend, zeigt die Arbeit, die erfolgreiche Synthese von Aspartatprotease-Sonden und deren Anwendung zur Identifikation von Interaktionspartnern einschließlich der exakten Interaktionsstelle. Allerdings sind weitere Optimierungen erforderlich, um eine breitere Anwendung zu ermöglichen. Wir schlagen eine Reihe möglicher Folgeexperimente vor und diskutieren zukünftige Perspektiven in Kapitel 6

A computational perspective of influenza a virus targets : neuraminidase and endonuclease.

Ph. D. University of KwaZulu-Natal, Durban 2016.Through the ages the viruses have plagued mankind claiming the lives of millions, pre-dating any advancements in the medicinal sciences. One such pathogenic virus is influenza A, which has been implicated in the 1918-Spanish flu, the 2006-avian flu outbreak and the 2009-swine flu pandemic. It is a highly sophisticated species, alluding efforts to thwart the spread of disease and infection. One of the main reasons influenza has survived this long is simple evolution. Natural mutation within the genome of virions expressed in proteins, enzymes or molecular structure render us unable to predict or take preventative measures against possible infection. Thus, research efforts toward the competitive inhibition of biological pathways that lead to the spread of disease, have become attractive targets. The influenza A virus has a number of chemotherapeutic targets, such as: 1) The surface antigens, hemagglutinin and neuraminidase, 2) RNA-dependent RNA polymerase, and 3) The M2 proton channel. Influenza RNA polymerase is composed of three large segments encoding polymerase acidic protein (PA), polymerase basic protein 1 (PB1) and polymerase basic protein 2 (PB2). The PA protein is an N-terminal domain subunit which contains the endonuclease activity. The influenza virus is incapable of synthesizing a 5’-mRNA cap, so it has adapted a cap-snatching mechanism whereby the PB2 subunit binds to the 5’-end of host mRNA, after which 10-14 nucleotides downstream the PA-subunit (aka PAN) cleaves the strand forming a primer for viral mRNA synthesis which is catalysed by the PB1 subunit. Influenza target identification is based primarily on evidence suggesting sequence conservation of each entity and its selective expression in the virus and not the host. In this thesis two enzymatic targets were investigated, the PA protein of RNA polymerase and neuraminidase. The studies focussed on using computational tools to: 1) provide insight into the mechanism of drug-resistance, 2) describe the conformational structure of the protein in the presence of point mutations and in complex with an inhibitor, 3) determine the essential binding pharmacophoric features to aid the design of new drug therapies. An array of computational techniques were employed in the studies, such as: molecular dynamics (MD) simulation, structure-based and ligand-based in silico screening, principal component analysis, radius of gyration analysis, binding free energy calculations and solventaccessible surface area analysis. The first study (Chapter 5) determined the mechanism of drug-resistance in influenza A neuraminidase as a consequence of antigenic variations. Two distinct mutations in the enzyme sequence that were investigated are H274Y and I222K. The active site residues of neuraminidase are conserved among the subtypes of influenza A. However, it was discovered that the occurrence of resistance to the drug oseltamivir, in the H1N1 species was different to the H5N1 virus. Although both systems shared a loss in hydrophobicity of the active site, the conformational distortion of the active site pocket distinguished the enzyme of the two viral entities, from one another. The discoveries made in the first study laid the foundation for the second study (Chapter 6), which was based on the in silico design and screen of potential neuraminidase inhibitors. As a result 10 characteristic molecular scaffolds were suggested as potential inhibitors. The pharmacophore design was constructed with consideration to the new conformational structure of the active site pocket. Chapter 7 is the third study of this thesis. The active site pocket enclosing the endonuclease activity of the PA subunit was investigated. Using molecular dynamics simulations and postdynamic analyses, a description of the protein conformation was offered. Subsequently, a pharmacophore was proposed as a potential scaffold to which endonuclease inhibitors may be modelled upon. It is my belief that the impact of the results derived from the above mentioned studies would greatly contribute to the development of new and effective anti-influenza drugs

Mechanism-based discovery of new anti-diabetic drugs from the natural products in traditional Chinese medicine

Insulin resistance is a fundamental metabolic defect of type 2 diabetes. Excessive lipid accumulation in the muscle and liver is the most common cause of insulin resistance. Hence, reducing lipid levels in these tissues can potentially reverse insulin resistance as an effective approach for the treatment of type 2 diabetes. Although there are a number of anti-diabetic drugs in clinical use, the long-term effects of the current medications are still unsatisfactory. Natural products have been recently rejuvenated as an important source for the discovery of anti-diabetic drugs due to their rich source and chemical diversity. Traditional Chinese Medicine has been used for centuries to treat diseases including diabetes. In order to identify the compounds with potential anti-diabetic properties by reducing lipid accumulation, a novel biochemical screening assay was developed using 3T3-L1 adipocytes and triglyceride content was assessed as readout of cellular lipids level. By using this assay, this thesis has screened >200 candidates selected from Traditional Chinese Medicine and identified 76 hits from >10 different classes in terms of triglyceride reduction. In the first animal study, the efficacy of albiflorin was tested in insulin resistant mice induced by high-fat cholesterol diet. Albiflorin significantly reduced triglyceride/cholesterol levels in the liver of high-fat cholesterol fed mice. Interestingly, further examinations of liver samples showed significant reduction of pro-inflammatory cytokine TNFα by albiflorin treatment. These results suggest albiflorin may have potential therapeutics for hepatic steatosis and associated liver metabolic conditions. To further test the effects of albiflorin on insulin resistance, this thesis examined its efficacy in a more established insulin resistant mice model induced by high-fat diet. Albiflorin significantly improved glucose intolerance in insulin resistant mice. However, further studies are required to confirm its therapeutic potential for insulin resistance. In the second animal study, this thesis investigated the effects oleanolic acid (OA) on hyperglycaemia in type 2 diabetic mice model. OA-treated mice effectively reduced hyperlipidaemia and reversed hyperglycaemia with liver as major target tissue. Moreover, this anti-hyperglycaemia effect was sustained beyond the treatment period. My subsequent studies focused on the mechanisms for the persistent effective maintenance of the corrected hyperglycaemia. By comparing the changes of key regulators in liver during and post-OA administration, this thesis found that the OA-induced changes in FoxO1, G6Pase, HAT1 and HDACs persisted in post-OA treatment where the increased phosphorylation of AMPK, SIRT1 and the reduced liver triglyceride had subsided. Further studies indicated that the anti-hyperglycaemic effects observed in post-OA treatment may result from persistent acetylation of FoxO1 to suppress hepatic gluconeogenic pathway. In summary, the results of my PhD project indicate using targeted screening approach for the intracellular lipid-lowering efficacy is an effective phenotypic screening tool, which is capable of identifying potential anti-obese and anti-diabetic compounds via different metabolisms. The present study highlights that mechanism-based discovery of compounds derived from our unique natural products library in Traditional Chinese Medicine have potential for the treatment of type 2 diabetes by reversing lipid-induced insulin resistance

The anti-proliferative activity of drimia altissima and a novel isolated flavonoid glycoside against hela cervical cancer cells

Cancer is one of the leading causes of mortality worldwide. About 44% of all cancer morbidity and 53% of all cancer mortality occur in countries with a low to medium Human Development Index (HDI). Thus, cancer is rapidly emerging as a serious threat to public health in Africa and most especially, sub-Saharan Africa. The International Agency for Research on Cancer (IARC) projects that there will be 1.28 million new cancer cases and 970 000 cancer deaths in Africa by the year 2030 owing to the increase in economic development associated lifestyles. The dominant types of cancer in Africa are those related to infectious diseases such as Kaposi’s sarcoma and cervical, hepatic and urinary bladder carcinomas. The main challenge to cancer treatment in Africa is the unavailability of efficacious anticancer drugs. This is because most developing countries can only afford to procure the most basic anticancer drugs, which are also frequently unavailable due to intermittent supplies. This results in patients progressing to more advanced cancer states. One way of combating this African problem is to focus on research that aims at discovering efficacious and cost effective cancer therapies from available natural resources within the African continent. This study investigated the potential anti-proliferative activity (against HeLa cervical cancer cells) of four plants (Adansonia digitata, Ceiba pentandra, Maytenus senegalensis and Drimia altissima) commonly used in the African traditional treatment of malignancies. After in vitro bio-assay screening using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, M. senegalensis root extract (MS-R) and D. altissima bulb extract (DA-B) showed anti-proliferative activity against HeLa cervical cancer cells with IC50 values of 25 μg/mL and 1.1 μg/mL respectively. By possessing the strongest anti-proliferative activity among the tested extracts, D. altissima was selected for further studies. Liquid-liquid partitioning of the Drimia altissima bulb extract with n-hexane, ethyl acetate, and n-butanol, yielded partitions 79a – d, with the n-butanol fraction, 79d, exhibiting the strongest cytotoxic activity (IC50 = 0.497 μg/mL). Through High Content Analysis (HCA) screening, fraction 79d was found to induce marked early mitotic cell cycle arrest. Fractionation of 79d using Diaion® HP-20 open column chromatography and a stepwise gradient of reducing polarity (water-methanol-ethanol-ethyl acetate) yielded cytotoxic fractions 82b, 82c, 82d and 82e, all with significant anti-proliferative activities at the tested concentrations of 0.1, 1.0 and 10 μg/mL. Bio-assay guided fractionation of 82c (the most effective fraction at the lowest tested concentration of 0.1 μg/mL) using Sephadex® LH-20 open column chromatography and 50% MeOH led to the isolation of compound 3.17. After structural elucidation using 1D and 2D Nuclear Magnetic Resonance spectroscopy (NMR), High resolution Mass spectrometry (HRMS), Fourier-Transform Infrared spectroscopy (FT-IR), ultraviolet spectroscopy (UV) and Circular Dichroism (CD), compound 3.17 was identified as a novel C-glucosylflavonoid-O-glucoside, 6-C-[-apio-α-D-furanosyl-(1→6)-β-glucopyranosyl]-4′, 5, 7-trihydroxyflavone (Altissimin, 3.17). Compound 3.17 exhibited a dose dependant anti-proliferative activity with an IC50 of 2.44 μM. The mechanism of action for compound 3.17 was investigated through cell cycle arrest, phosphatidylserine translocation (PS), caspase activation and mitochondrial membrane depolarization. The mechanism of cell death elicited by compound 3.17 in HeLa cells was found to involve the induction of M phase cell cycle arrest with consequent activation of apoptotic cell death which was evident from annexin V staining, mitochondrial membrane potential (ΔΨm) collapse and the activation of caspases -8 and -3. In silico computational techniques were employed to virtually determine potential biological targets of compound 3.17. Target fishing using the Similarity Ensemble Approach (SEA) target prediction gave human aldose reductase (hAR, AKR1B1) the highest ranking with a p value of 2.85 x 10-24, a max Tc of 0.35 and a Z-score of 41.8217. Using AutoDock4 and the AutoDock tools suite (ADT), molecular docking of compound 3.17 in the hAR binding pocket was successfully achieved with a lower ΔG free energy binding (-9.4 kcal/mol) than that of positive control ligand 393 (-8.7 kcal/mol). In conclusion, this study identified the genus Drimia and particularly D. altissima as a potential source for novel cytotoxic compounds. The discovery of altissimin (3.17), the first flavonoid glycoside to be isolate from D. altissima, enquires into the possible existence of similar compounds within the species. In addition to the observed in vitro cytotoxic activity against HeLa cells, the potential of altissimin (3.17) as a hAR enzyme inhibitor opens up the possibility of its use as an adjunct to increase cancer cell sensitivity to chemotherapy. Thus, altissimin (3.17) shows promise as a potential anticancer agent