Design, synthesis and biological evaluation of novel thiadiazoline-thiazolone hybrids as kinase inhibitors.

Masters Degree. University of KwaZulu-Natal, Durban. 2017Cancer is a leading cause of death globally, and it was responsible for 8.8 million deaths in 2015. It is predicted that there will be 22 million new cancer cases by 2030 worldwide. Approximately, 70% of deaths from cancer occur in low- and middle-income countries. Furthermore, breast cancer is the second most common cancer among South African women and is reported to affect 1 in every 26 women. The social and economic burdens associated with cancers are severe at national and international levels hence, there is an urgent need for the development of more effective cancer therapeutics. To accomplish this aspect, in this study, thiadiazole-thiazolone (TDT) hybrids were developed as dual inhibitors of cyclindependent kinase (CDK) and kinesin spindle protein (KSP), respectively. Twenty-two novel TDT hybrid compounds (8a-v) were synthesized using multistep organic synthesis and were characterized using thin layer chromatography (TLC), infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (1H and 13C NMR), and high-resolution mass spectrometry (HR-MS). All the compounds (8a-v) were screened for their potential in vitro inhibition of validated anticancer drug targets (CDK, Abl and KSP) and cancer cell lines (MCF-7 and K562). Results obtained from these evaluations suggested that the synthesized compounds were potent inhibitors of CDK and KSP thus confirming the dual mode of action. Amongst, 8h was identified as the most potent compound with an IC50 value of 3.1 µM against CDK2 enzyme and exhibited good cytotoxicity (GI50 = 6.25 µM) against the tested cancer cell lines (MCF-7 and K-562). A brief structure-activity relationship (SAR) analysis indicated that 2- chloro and 4-nitro substituents on the phenyl ring of the thiazolone motif contributed significantly to the inhibition of both of the anticancer drug targets (CDK and KSP). An in silico molecular docking study using the crystal structures of the target enzymes (CDK-2 and KSP) further supported the SAR and extrapolated the importance of crucial molecular interactions in influencing the enzyme inhibitory activitie

Exploring the Role of Molecular Dynamics Simulations in Most Recent Cancer Research: Insights into Treatment Strategies

Cancer is a complex disease that is characterized by uncontrolled growth and division of cells. It involves a complex interplay between genetic and environmental factors that lead to the initiation and progression of tumors. Recent advances in molecular dynamics simulations have revolutionized our understanding of the molecular mechanisms underlying cancer initiation and progression. Molecular dynamics simulations enable researchers to study the behavior of biomolecules at an atomic level, providing insights into the dynamics and interactions of proteins, nucleic acids, and other molecules involved in cancer development. In this review paper, we provide an overview of the latest advances in molecular dynamics simulations of cancer cells. We will discuss the principles of molecular dynamics simulations and their applications in cancer research. We also explore the role of molecular dynamics simulations in understanding the interactions between cancer cells and their microenvironment, including signaling pathways, proteinprotein interactions, and other molecular processes involved in tumor initiation and progression. In addition, we highlight the current challenges and opportunities in this field and discuss the potential for developing more accurate and personalized simulations. Overall, this review paper aims to provide a comprehensive overview of the current state of molecular dynamics simulations in cancer research, with a focus on the molecular mechanisms underlying cancer initiation and progression.Comment: 49 pages, 2 figure

Correlation between cell line chemosensitivity and protein expression pattern as new approach for the design of targeted anticancer small molecules

BACKGROUND AND RATIONALE: Over the past few decades, several databases with a significant amount of biological data related to cancer cells and anticancer agents (e.g.: National Cancer Institute database, NCI; Cancer Cell Line Encyclopedia, CCLE; Genomic and Drug Sensitivity in Cancer portal, GDSC) have been developed. The huge amount of heterogeneous biological data extractable from these databanks (among all, drug response and protein expression) provides a real foundation for predictive cancer chemogenomics, which aims to investigate the relationships between genomic traits and the response of cancer cells to drug treatment with the aim to identify novel therapeutic molecules and targets. In very recent times many computational and statistical approaches have been proposed to integrate and correlate these heterogeneous biological data sequences (protein expression – drug response), with the aim to assign the putative mechanism of action of anticancer small molecules with unknown biological target/s. The main limitation of all these computational methods is the need for experimental drug response data (after screening data). From this point of view, the possibility to predict in silico the antiproliferative activity of new/untested small molecules against specific cell lines, could enable correlations to be found between the predicted drug response and protein expression of the desired target from the very earliest stages of research. Such an innovative approach could allow to select the compounds with molecular mechanisms that are more likely to be connected with the target of interest preliminary to the in vitro assays, which would be a critical aid in the design of new targeted anticancer agents. RESULTS: In the present study, we aimed to develop a new innovative computational protocol based on the correlation of drug activity and protein expression data to support the discovery of new targeted anticancer agents. Compared with the approaches reported in the literature, the main novelty of the proposed protocol was represented by the use of predicted antiproliferative activity data, instead of experimental ones. To this aim, in the first phase of the research the new in silico Antiproliferative Activity Predictor (AAP) tool able to predict the anticancer activity (expressed as GI50) of new/untested small molecules against the NCI-60 panel was developed. The ligand-based tool, which took the advantages of the consolidated expertise of the research group in the manipulation of molecular descriptors, was adequately validated and the reliability of the prediction was further confirmed by the analysis of an in-house database and subsequent evaluation of a set of molecules selected by the NCI for the one-dose/five-doses antiproliferative assays. In the second part of the study, a new computational method to correlate drug activity data and protein expression pattern data was proposed and evaluated by analysing several case studies of targeted drugs tested by NCI, confirming the reliability of the proposed method for the biological data analysis. In the last part of the project the proposed correlation approach was applied to design new small molecules as selective inhibitors of Cdc25 phosphatase, a well-known protein involved in carcinogenic processes. By means of this innovative approach, integrated with other classical ligand/structures-based techniques, it was possible to screen a large database of molecular structures, and to select the ones with optimal relationship with the focused target. In vitro antiproliferative and enzymatic inhibition assays of the selected compounds led to the identification of new structurally heterogeneous inhibitors of Cdc25 proteins and confirmed the results of the in silico analysis. CONCLUSIONS: Collectively, the obtained results showed that the correlation between protein expression pattern and chemosensitivity is an innovative, alternative, and effective method to identify new modulators for the selected targets. In contrast to traditional in silico methods, the proposed protocol allows for the selection of molecular structures with heterogeneous scaffolds, which are not strictly related to the binding sites and with chemical-physical features that may be more suitable for all the pathways involved in the overall mechanism. The biological assays further corroborate the robustness and the reliability of this new approach and encourage its application in the anticancer targeted drug discovery field

Design, synthesis and pharmacological evaluation of novel fused pyrimidine analogues as anticancer agents.

Doctoral Degree. University of KwaZulu-Natal, Durban.Cancer is a multifaceted disease considered as the most serious health burden all over the world. Due to existing of limited anticancer drugs and detrimental side effects, the anticancer research has been challenging. An investigation on identifying novel potential drugs is highly required to treat this serious abnormal cell growth. Advanced potential anticancer drug entrants are crucially required to combat the drawbacks linked with current drugs or line of therapies. Extensive investigations are being carried out on synthetic manipulations of heterocyclic aromatic compounds (purines) for developing efficient and potent anticancer drugs. Besides, these manipulations also offer effective leads for further optimization. Therefore, this project is an effort in detecting a novel and potent anticancer leads based on bioisostere of purines called pyrazolopyrimidines. In this research project we have performed an comprehensive literature survey of structural isomers of pyrazolopyrimidines (pyrazolo[1,5-a]pyrimidine and pyrazolo[4,3-d]pyrimidine) for their synthetic approaches and biological activities with special emphasis on structure-activity relationship (SAR) studies. These SAR studies prompted us to implement the observed studies on one of the structural isomer of pyrazolopyrimidine called pyrazolo[3,4-d]pyrimidine. And further, we have synthesized some novel series of pyrazolo[3,4-d]pyrimidine derivatives with various substituents at C-4 and C-6 positions of the scaffold. A total 71 compounds comprising of phenethyl and pentane hybrids (7-43, Chapter 4), benzoyl hybrids (5a-5h, 6a-6d and 7a-7c, Chapter 5) and lastly phenylcarbamoyl acetamide hybrids (9a-9s, Chapter 6) have been synthesized by molecular hybridization approach as outlined in schemes of respective chapters. The completion of reaction and the purity of novel synthesized compounds were confirmed by chromatographic analysis. All the newly synthesized compounds displayed acceptable analysis for their anticipated structures, which were established based on physicochemical and spectral data (IR, 1 H NMR, 13C NMR and HRMS). All synthesized compounds were primarily evaluated for their in vitro anticancer activities at Laboratory of Growth Regulators, Centre of the Region Hana for Biotechnological and Agricultural Research, Palacky University & Institute of Experimental Botany ASCR, Slechtitelu 27, 78371 Olomouc, Czech Republic. From the systematic analysis of anticancer activity, results obtained following key observations were made. i. Structural isomers of fused pyrimidines have been looked upon for molecular changes in emerging drug like candidates. Pyrazolopyrimidine is a bioisostere of purines has acquired considerable importance due to its diverse, facile and general synthetic methodologies with great medicinal importance. Several analogs of this scaffold have emerged as a promising leads in the design of some novel pharmacologically active compounds with enhanced iii metabolic, pharmacokinetic and pharmacological profiles, representing that there is plenty scope for considering pyrazolopyrimidine as a structural framework for evolving effective leads. ii. Chapter 4: From the 37 novel phenethyl and alkyl pentane pyrazolo[3,4-d]pyrimidine derivatives synthesized and evaluated for CDK2/Cyclin E, Abl kinase inhibitory activity and anti-proliferative activity against K-562 (chronic myelogeneous leukemia) and MCF7 (breast adenocarcinoma) cell lines. From the tested results, compounds 11 (CDK: IC50 = 5.1 µM; Abl: ˃12.5 µM), 8 (CDK: IC50 = 7.8 µM; Abl: ˃25 µM) and 36 (CDK: IC50 = 8.8 µM; Abl: >25 µM) exhibited significant inhibitory activity. Further from this series, most of the synthesized compounds indicated prominent anti-proliferative effects with IC50 value ranging from 19.2 µM to 27.4 µM. Incorporation of monosubstituted phenyl groups at C-4 of the pyrazolo[3,4-d]pyrimidine nucleus had favored for most prominent anticancer activity. iii. Chapter 5: Among the 15 novel benzoyl hybrids synthesized and evaluated, compounds 5a and 6c displayed (CDK2: IC50 = 8.8 µM, 6.8 µM) commendable inhibitory activity and notable anti-proliferative activity ranging from 18.9 µM to 89.3 µM). Presence of heteroatom containing bicyclic moieties at C-4 of the nucleus enhanced both inhibitory and anti-proliferative activity. iv. Chapter 6: Of the 19 novel phenylcarbamoyl acetamide hybrids synthesized and tested, compounds 9a, 9c, 9g, 9m and 9p showed moderate enzymatic inhibitory activity with an IC50 value ˃12.5 µM against both CDK2 and Abl kinases while, remaining compounds of this series could not generate IC50 values due to solubility limit (IC50 = ˃25 µM to ˃100 µM).Professor Neil Koorbanally was acknowledged by the author as his previous supervisor

Small molecules containing chalcogen elements (S, Se, Te) as new warhead to fight neglected tropical diseases

Neglected tropical diseases (NTDs) encompass a group of infectious diseases with a protozoan etiology, high incidence, and prevalence in developing countries. As a result, economic factors constitute one of the main obstacles to their management. Endemic countries have high levels of poverty, deprivation and marginalization which affect patients and limit their access to proper medical care. As a matter of fact, statistics remain un- collected in some affected areas due to non-reporting cases. World Health Organization and other organizations proposed a plan for the eradication and control of the vector, although many of these plans were halted by the COVID-19 pandemic. Despite of the available drugs to treat these pathologies, it exists a lack of effectiveness against several parasite strains. Treatment protocols for diseases such as American trypanosomiasis (Chagas disease), leishmaniasis, and human African trypanosomiasis (HAT) have not achieved the desired results. Un- fortunately, these drugs present limitations such as side effects, toxicity, teratogenicity, renal, and hepatic impairment, as well as high costs that have hindered the control and eradication of these diseases. This review focuses on the analysis of a collection of scientific shreds of evidence with the aim of identifying novel chalcogen- derived molecules with biological activity against Chagas disease, leishmaniasis and HAT. Compounds illustrated in each figure share the distinction of containing at least one chalcogen element. Sulfur (S), selenium (Se), and tellurium (Te) have been grouped and analyzed in accordance with their design strategy, chemical synthesis process and biological activity. After an exhaustive revision of the related literature on S, Se, and Te compounds, 183 compounds presenting excellent biological performance were gathered against the different causative agents of CD, leishmaniasis and HAT

Development of Targeted Therapeutics for the Treatment of Glioblastoma

Glioblastoma is the most aggressive cancer of the brain. Despite recent advances in cancer biology and multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome of patients with high grade glioma remains fatal. The major drawback of current glioma chemotherapeutics is their inability to cross the blood brain barrier, lack of tumour specificity agents and their consequent side effects. Matrix metalloprotease (MMP) activity is central to cancer development, angiogenesis and invasion. They are highly active in the tumour environment and absent or inactive in normal tissues, therefore they represent viable targets for cancer drug discovery. A better understanding of the role of MMPs in human gliomas could potentially have diagnostic, prognostic and therapeutic implications. This study aims to assess the expression of specific MMPs in preclinical human glioma models and clinical glioma samples; evaluate in silico docking to rationalise substrate binding preferences of homologous MMPs; rationally design MMP-subtype-selective tumour activated prodrugs; and determine the feasibility of targeting MMP-selective anticancer prodrugs conjugated to graphene oxide as a local drug delivery approach for glioblastoma. This study found significant overexpression of MMP-10 in glioma relative to histologically normal brain tissues. Strong correlation was observed between MMP-10 protein and gene expression of glioma cell lines relative to low expression in a normal brain cell line. MMP-10 activity, as measured by fluorogenic substrate cleavage assay, also demonstrated a strong correlation between MMP-10 activity and gene expression levels. Following demonstration of selective overexpression of MMP-10 in glioma, a reiterative in silico proteolytic docking coupled in vitro biochemical assessment was utilised to rationalise functional similarity and differentiate substrate binding selectivity of homologous MMPs. The binding modes of MMP-substrates within the active site of closely related MMPs were able to accurately predict the cleavage subsites by specific MMPs, as confirmed by in vitro cleavage assay. The success of computational and experimental methodology provided a robust tool for identifying MMP-subtype differences and subsequent development of MMP-10 selective peptide prodrugs. MMP-subtype selective and MMP-10 selective prodrugs were designed by rational exploitation of MMP-docked complexes of substrates. Peptide residues were modified to achieve selectivity for MMP-2 and MMP-10 (over MMP-3 and MMP-9) demonstrating predicted cleavage at distinct subsites. This selectivity was further exploited to attain MMP-10 selectivity, over MMP-2, MMP-3 and MMP-9. The rationally designed peptide prodrugs were synthesised and were shown to be preferentially cleaved by MMPs at predicted subsites and demonstrated no activation by engineered-out MMPs, as predicted. Compared to MJ02 (MMP-2 and MMP-10 selective doxorubicin prodrug), MJ04 (MMP-10 selective doxorubicin prodrug) demonstrated selective metabolism by glioma cell lines to release chemotherapeutic agents. This therapeutic approach against glioma cell lines depended upon the involvement of MMPs, confirmed using pharmacological inhibition. MJ04 demonstrated negligible activity in the presence of an MMP-10 selective inhibitor, suggesting MMP-10 selective activation of the prodrug in glioma cells relative to normal glial cells. Following successful development of MMP-10 selective prodrugs, the feasibility of targeting glioma tumour with local delivery of chemotherapeutics from functionalised graphene-oxide tethered prodrug implants, was assessed as a therapeutic strategy to circumvent the blood brain barrier. Graphene oxide conjugated prodrug was synthesised which is shown to be preferentially cleaved in MMP expressing glioma cell lines relative to normal glial cells. This study demonstrates that MMP-10 is overexpressed in glioblastoma and can be used to metabolise anticancer prodrugs that can be activated selectively by local tumour environment

Dosing-time makes the poison : circadian regulation and pharmacotherapy

Daily rhythms in physiology significantly modulate drug pharmacokinetics and pharmacodynamics according to the time-of-day, a finding that has led to the concept of chronopharmacology. The importance of biological clocks for xenobiotic metabolism has gained increased attention with the discovery of the molecular circadian clockwork. Mechanistic understanding of the cell-autonomous molecular circadian oscillator and the circadian timing system as a whole has opened new conceptual and methodological lines of investigation to understand first, the clock's impact on a specific drug's daily variations or the effects/side effects of environmental substances, and second, how clock-controlled pathways are coordinated within a given tissue or organism. Today, there is an increased understanding of the circadian modulation of drug effects. Moreover, several molecular strategies are being developed to treat disease-dependent and drug-induced clock disruptions in humans

Structural characterization and selective drug targeting of higher-order DNA G-quadruplex systems.

There is now substantial evidence that guanine-rich regions of DNA form non-B DNA structures known as G-quadruplexes in cells. G-quadruplexes (G4s) are tetraplex DNA structures that form amid four runs of guanines which are stabilized via Hoogsteen hydrogen bonding to form stacked tetrads. DNA G4s have roles in key genomic functions such as regulating gene expression, replication, and telomere homeostasis. Because of their apparent role in disease, G4s are now viewed as important molecular targets for anticancer therapeutics. To date, the structures of many important G4 systems have been solved by NMR or X-ray crystallographic techniques. Small molecules developed to target these structures have shown promising results in treating cancer in vitro and in vivo, however, these compounds commonly lack the selectivity required for clinical success. There is now evidence that long single-stranded G-rich regions can stack or otherwise interact intramolecularly to form G4-multimers, opening a new avenue for rational drug design. For a variety of reasons, G4 multimers are not amenable to NMR or X-ray crystallography. In the current dissertation, I apply a variety of biophysical techniques in an integrative structural biology (ISB) approach to determine the primary conformation of two disputed higher-order G4 systems: (1) the extended human telomere G-quadruplex and (2) the G4-multimer formed within the human telomerase reverse transcriptase (hTERT) gene core promoter. Using the higher-order human telomere structure in virtual drug discovery approaches I demonstrate that novel small molecule scaffolds can be identified which bind to this sequence in vitro. I subsequently summarize the current state of G-quadruplex focused virtual drug discovery in a review that highlights successes and pitfalls of in silico drug screens. I then present the results of a massive virtual drug discovery campaign targeting the hTERT core promoter G4 multimer and show that discovering selective small molecules that target its loops and grooves is feasible. Lastly, I demonstrate that one of these small molecules is effective in down-regulating hTERT transcription in breast cancer cells. Taken together, I present here a rigorous ISB platform that allows for the characterization of higher-order DNA G-quadruplex structures as unique targets for anticancer therapeutic discovery

IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD