Repurposing of US-FDA-approved drugs as negative modulators of ubiquitin specific protease-7 (USP7).

Ubiquitin-specific protease7 (USP7) regulates the stability of the p53 tumor suppressor protein and several other proteins critical for tumor cell survival. Aberrant expression of USP7 facilitates human malignancies by altering the activity of proto-oncogenes/proteins, and tumor suppressor genes. Therefore, USP7 is a validated anti-cancer drug target. In this study, a drug repurposing approach was used to identify new hits against the USP7 enzyme. It is one of the most strategic approaches to find new uses for drugs in a cost- and time-effective way. Nuclear Magnetic Resonance-based screening of 172 drugs identified 11 compounds that bind to the catalytic domain of USP7 with dissociation constant (Kd) values in the range of 0.6-1.49 mM. These 11 compounds could thermally destabilize the USP7 enzyme by decreasing its melting temperature up to 9 °C. Molecular docking and simulation studies provided structural insights into the ligand-protein complexes, suggesting that these compounds bind to the putative substrate binding pocket of USP7, and interact with its catalytically important residues. Among the identified 11 hits, compound 6 (oxybutynin), 7 (ketotifen), 10 (pantoprazole sodium), and 11 (escitalopram) also showed anti-cancer activity with an effect on the expression of proto-oncogenes and tumor-suppressor gene at mRNA level in HCT116 cells. The compounds identified in this study can serve as potential leads for further studies.

Drugs repurposing: An approach to identify new hits against anticancer drug target TFIIH subunit p8.

Drug repositioning is one of the most effective approaches towards drug discovery and development. It involves the identification of new therapeutic indications of existing drugs. The present study evaluated several drugs for their ability to modulate activity of the p8 subunit of TFIIH complex. Negative modulation of p8 subunit activity disrupts protein-protein interactions (PPIs) among the subunits of TFIIH complex, and thereby the TFIIH-associated functions. TFIIH complex has key role in the transcription and nucleotide excision repair activity in cancerous cells. TFIIH complex has emerged as a privileged drug target in anticancer research. Out of 60 drugs, amlopipine (13), diltiazem (16), gemfibrozil (19), levocitrizine dihydrochloride (20), losartan potassium (22), clorthalidone (24), and escitalopram (28) showed interactions with subunit p8 in the ligand-protein binding and chemical shift perturbation studies. The Kd values were found to be between 0.25 and 1 mM. These drugs also caused thermal destabilization of the subunit p8 by negatively shifting the melting temperature by ≥ 2 °C. Molecular docking studies indicated the interaction of these drugs with important residues of p8-p52 complex, such as Glu48, Lys51, Glu496, and Glu455 via non-covalent interactions. This study has thereby identified 7 drugs that can be investigated further as potential anticancer drugs.

Identification of new lead molecules against anticancer drug target TFIIH subunit P8 using biophysical and molecular docking studies.

The identification of molecules, which could modulate protein-protein interactions (PPIs), is of primary interest to medicinal chemists. Using biophysical methods during the current study, we have screened 76 compounds (grouped into 16 mixtures) against the p8 subunit of the general transcription factor (TFIIH), which has recently been validated as an anti-cancer drug target. 10% of the tested compounds showed interactions with p8 protein in STD-NMR experiments. These results were further validated by molecular docking studies where interactions between compounds and important amino acid residues were identified, including Lys20 in the hydrophobic core of p8, and Asp42 and 43 in the ß3 strand. Moreover, these compounds were able to destabilize the p8 protein by negatively shifting the Tm (≥2 °C) in thermal shift assay. Thus, this study has identified 8 compounds which are likely negative modulators of p8 protein stability, and could be further considered as potential anticancer agents.

In-vitro and in-vivo anticandidal activity of Trachyspermum ammi (L.) sprague seeds ethanolic extract and thymol-containing hexanes fraction.

Candida albicans is the causative agent of candidiasis in immunocompromised and diseased individuals. The current study was designed to evaluate the antifungal activity of Trachyspermum ammi (L.) Sprague seeds ethanolic extract and hexanes fraction against Candida albicans, both in-vitro and in-vivo. The minimal inhibitory concentration of hexanes fraction was found to be 225 µg/mL, as compared to the standard drug amphotericin B (200 µg/mL). For the treatment of cutaneous candidiasis in BALB/c mice model, the extract and fraction containing ointment was topically applied on flank of mice at various concentrations, and 90-100% recovery in mice was observed which was better than the standard drug, clotrimazole. The GC/MS analysis of hexanes extract indicated the presence of three major components, including (43.91%), O-cymene (25.53%), and γ-terpinene (22.64%). The results showed that T. ammi possess potential antifungal effect, and can be used for the development of novel antifungal agents after further pre-clinical and clinical trials.

Thymidine phosphorylase and prostrate cancer cell proliferation inhibitory activities of synthetic 4-hydroxybenzohydrazides: In vitro, kinetic, and in silico studies.

Over-expression of thymidine phosphorylase (TP) plays a key role in many pathological complications, including angiogenesis which leads to cancer cells proliferation. Thus in search of new anticancer agents, a series of 4-hydroxybenzohydrazides (1-29) was synthesized, and evaluated for in vitro thymidine phosphorylase inhibitory activity. Twenty compounds 1-3, 6-14, 16, 19, 22-24, and 27-29 showed potent to weak TP inhibitory activities with IC50 values in the range of 6.8 to 229.5 µM, in comparison to the standards i.e. tipiracil (IC50 = 0.014 ± 0.002 µM) and 7-deazaxanthine (IC50 = 41.0 ± 1.63 µM). Kinetic studies on selected inhibitors 3, 9, 14, 22, 27, and 29 revealed uncompetitive and non-competitive modes of inhibition. Molecular docking studies of these inhibitors indicated that they were able to interact with the amino acid residues present in allosteric site of TP, including Asp391, Arg388, and Leu389. Antiproliferative (cytotoxic) activities of active compounds were also evaluated against mouse fibroblast (3T3) and prostate cancer (PC3) cell lines. Compounds 1, 2, 19, and 22-24 exhibited anti-proliferative activities against PC3 cells with IC50 values between 6.5 to 10.5 µM, while they were largely non-cytotoxic to 3T3 (mouse fibroblast) cells proliferation. Present study thus identifies a new class of dual inhibitors of TP and cancer cell proliferation, which deserves to be further investigated for anti-cancer drug development.

Natural compounds as angiogenic enzyme thymidine phosphorylase inhibitors: In vitro biochemical inhibition, mechanistic, and in silico modeling studies.

Natural flora is the richest source of novel therapeutic agents due to their immense chemical diversity and novel biological properties. In this regard, eighteen natural products belonging to different chemical classes were evaluated for their thymidine phosphorylase (TP) inhibitory activity. TP shares identity with an angiogenic protein platelet derived endothelial cell growth factor (PD-ECGF). It assists tumor angiogenesis and is a key player in cancer progression, thus an ideal target to develop anti-angiogenic drugs. Eleven compounds 1-2, 5-10, 11, 15, and 18 showed a good to weak TP inhibitory activity (IC50 values between 44.0 to 420.3 µM), as compared to standards i.e. tipiracil (IC50 = 0.014 ± 0.002 µM) and 7-deazaxanthine (IC50 = 41.0 ± 1.63 µM). Kinetic studies were also performed on active compounds, in order to deduce the mechanism of ligand binding to enzyme. To get further insight into receptor protein (enzyme) and ligand interaction at atomic level, in- sillico studies were also performed. Active compounds were finally evaluated for cytotoxicity test against mouse fibroblast (3T3) cell line. Compound 18 (Masoprocol) showed a significant TP inhibitory activity (IC50 = 44.0 ± 0.5 µM). Kinetic studies showed that it inhibits the enzyme in a competitive manner (Ki = 25.6 ± 0.008 µM), while it adopts a binding pose different than the substrate thymidine. It is further found to be non-toxic in MTT cytotoxicity assay. This is the first report on TP inhibitory activity of several natural compounds, some of which may serve as leads for further research towards drug the development.

Small molecule-based targeting of TTD-A dimerization to control TFIIH transcriptional activity represents a potential strategy for anticancer therapy.

The human transcription factor TFIIH is a large complex composed of 10 subunits that form an intricate network of protein-protein interactions critical for regulating its transcriptional and DNA repair activities. The trichothiodystrophy group A protein (TTD-A or p8) is the smallest TFIIH subunit, shuttling between a free and a TFIIH-bound state. Its dimerization properties allow it to shift from a homodimeric state, in the absence of a functional partner, to a heterodimeric structure, enabling dynamic binding to TFIIH. Recruitment of p8 at TFIIH stabilizes the overall architecture of the complex, whereas p8's absence reduces its cellular steady-state concentration and consequently decreases basal transcription, highlighting that p8 dimerization may be an attractive target for down-regulating transcription in cancer cells. Here, using a combination of molecular dynamics simulations to study p8 conformational stability and a >3000-member library of chemical fragments, we identified small-molecule compounds that bind to the dimerization interface of p8 and provoke its destabilization, as assessed by biophysical studies. Using quantitative imaging of TFIIH in living mouse cells, we found that these molecules reduce the intracellular concentration of TFIIH and its transcriptional activity to levels similar to that observed in individuals with trichothiodystrophy owing to mutated TTD-A Our results provide a proof of concept of fragment-based drug discovery, demonstrating the utility of small molecules for targeting p8 dimerization to modulate the transcriptional machinery, an approach that may help inform further development in anticancer therapies.

Synthesis, molecular docking and xanthine oxidase inhibitory activity of 5-aryl-1H-tetrazoles.

5-Aryl-1H-tetrazoles (1-24) were synthesized and screened for their xanthine oxidase (XO) inhibitory activity using allopurinol as standard inhibitor (IC50 = 2.0 ±â€¯0.01 µM). Six compounds 3, 4, 5, 9, 21, and 24 exhibited significant to weak activities with IC50 values in the range of 7.4-174.2 µM. Active compounds were further subjected to kinetic and molecular docking studies to deduce their modes of inhibition, and to study their interactions with the protein (XO) at atomic level, respectively. Interestingly, all these compounds showed a competitive mode of inhibition. Docking studies identified several important interactions between the ligand and the receptor protein (XO). Some of these interactions were similar to that exhibited by clinical inhibitors of XO (allopurinol, and febuxostat). This study identifies 5-aryl-1H-tetrazoles as a new class of xanthine oxidase inhibitors, which deserves to be further, investigated for the treatment of hyperuricemia and gout.

Xanthine Oxidase Inhibitory and Molecular Docking Studies on Pyrimidones.

BACKGROUND: Xanthine oxidase is an important enzyme which catalyzes the production of uric acid and superoxide anion from xanthine. The over-production of these products leads to different disease conditions. For instance, uric acid is responsible for hyperuricemia, gout, and arthritis, while superoxide anion contributes to the oxidative stress, and related diseases. Hence XO is an important pharmacological target for the treatment of a range of diseases. METHODS: Based on the structural resemblance of pyrimidines with xanthine, a series of previously synthesized ethyl 6- methyl-2-oxo-1, 2, 3, 4-tetrahydro-5-pyrimidinecarboxylate derivatives were evaluated for XO inhibitory activity. RESULTS: Among 25 pyrimidone derivatives, 22 were found to be good to weak inhibitors with IC50 values in the range of 14.4 - 418 µM. Compounds 3, 14, 15, 18, and 21-23 were significant inhibitors, and thus analyzed for their kinetic parameters. Among them compounds 14, 15, 18, and 23 were competitive, 21 and 22 showed non-competitive, while 23 was a mixed-type of inhibitor. Molecular docking studies highlighted the interactions of these inhibitors with critical amino acids of XO, such as Val1011, Phe649, Lys771, and others. Moreover, the cytotoxicity studies on these selected inhibitors showed all these compounds to be non-cytotoxic. CONCLUSION: These non-cytotoxic, significant XO inhibitors can thus be further investigated for the treatment of hyperuricemia, and gout.

Thymidine esters as substrate analogue inhibitors of angiogenic enzyme thymidine phosphorylase in vitro.

Thymidine phosphorylase (TP) catalyzes the cleavage of thymidine into thymine and 2-deoxy-α-d-ribose-1-phosphate. Elevated activity of TP prevents apoptosis, and induces angiogenesis which ultimately leads to tumor growth and metastasis. Critical role of TP in cancer progression makes it a valid target in anti-cancer research. Discovery of small molecules as TP inhibitors is vigorously pursued in cancer therapy. In the present study, we functionalized thymidine as benzoyl ester to synthesize compounds 3-16. In vitro evaluation of thymidine esters for their thymidine phosphorylase inhibition activity was subsequently carried out. Compounds 4, 10, 14, and 15 showed good activities with lower IC50 values than the standard, 7-deazaxanthine (IC50=41.0±1.63µM). Among them, compound 14 showed five folds higher activity (IC50=7.5±0.8µM), while 4 (IC50=18.5±1.0µM) and 10 (IC50=18.8±1.2µM) showed two folds higher activity than the standard. Compound 15 showed slightly better activity (IC50=33.3±1.5µM) to the standard. Potent compounds were further subjected to kinetic and molecular docking studies to identify their mode of inhibition, and to study their interactions with the protein at atomic level, respectively. All active compounds were non-cytotoxic to mouse fibroblast 3T3 cell line. These results identify thymidine esters as substrate analogue (substrate-like) inhibitors of angiogenic enzyme thymidine phosphorylase for further studies.

Unexpected central role of the androgen receptor in the spontaneous regeneration of myelin.

Lost myelin can be replaced after injury or during demyelinating diseases in a regenerative process called remyelination. In the central nervous system (CNS), the myelin sheaths, which protect axons and allow the fast propagation of electrical impulses, are produced by oligodendrocytes. The abundance and widespread distribution of oligodendrocyte progenitors (OPs) within the adult CNS account for this remarkable regenerative potential. Here, we report a key role for the male gonad, testosterone, and androgen receptor (AR) in CNS remyelination. After lysolecithin-induced demyelination of the male mouse ventral spinal cord white matter, the recruitment of glial fibrillary acidic protein-expressing astrocytes was compromised in the absence of testes and testosterone signaling via AR. Concomitantly, the differentiation of OPs into oligodendrocytes forming myelin basic protein (MBP)+ and proteolipid protein-positive myelin was impaired. Instead, in the absence of astrocytes, axons were remyelinated by protein zero (P0)+ and peripheral myelin protein 22-kDa (PMP22)+ myelin, normally only produced by Schwann cells in the peripheral nervous system. Thus, testosterone favors astrocyte recruitment and spontaneous oligodendrocyte-mediated remyelination. This finding may have important implications for demyelinating diseases, psychiatric disorders, and cognitive aging. The testosterone dependency of CNS oligodendrocyte remyelination may have roots in the evolutionary history of the AR, because the receptor has evolved from an ancestral 3-ketosteroid receptor through gene duplication at the time when myelin appeared in jawed vertebrates.

2-Arylquinazolin-4(3H)-ones: A novel class of thymidine phosphorylase inhibitors.

Thymidine phosphorylase (TP) over expression plays an important role in several pathological conditions, such as rheumatoid arthritis, chronic inflammatory diseases, psoriasis, and tumor angiogenesis. In this regard, a series of twenty-five 2-arylquinazolin-4(3H)-one derivatives 1-25 were evaluated for thymidine phosphorylase inhibitory activity. Six compounds 5, 6, 20, 2, 23, and 3 were found to be active against thymidine phosphorylase enzyme with IC50 values in the range of 42.9-294.6µM. 7-Deazaxanthine (IC50=41.0±1.63µM) was used as a standard inhibitor. Compound 5 showed a significant activity (IC50=42.9±1.0µM), comparable to the standard. The enzyme kinetic studies on the most active compounds 5, 6, and 20 were performed for the determination of their modes of inhibition, and dissociation constants Ki. All active compounds were found to be largely non-cytotoxic against the mouse fibroblast 3T3 cell line. This study identifies a novel class of thymidine phosphorylase inhibitors which may be further investigated as leads to develop therapeutic agents.

Zwitterionic pyrimidinium adducts as antioxidants with therapeutic potential as nitric oxide scavenger.

A variety of zwitterionic adducts were synthesized by using means green chemistry method. The products contain the biologically active barbituric acid moiety embedded in zwitterion products. Both features are pharmaceutically relevant. The chemical structures were deduced by (1)H-, (13)C-, NMR and HRMS spectral analysis, and X-ray diffraction techniques. In vitro evaluation for the antioxidant activities were carried out towards the inhibition of nitric oxide (NO) radical, known to regulate a mechanism of signals for various cellular functions. NO also play an important role as a mediator of various pathological conditions responsible for cellular damages such as strokes, cancers, diabetes, chronic heart failure and inflammatory disease and various neurodegenerative disorders. All tested compounds were found to be more potent nitric oxide scavengers as compared to standard drug ascorbic acid (IC50 = 618 ± 2.0 µM). Compounds 4c and e exhibiting several hundred fold more activity against nitric oxide radical with IC50 values of 69 ± 1.66 and 70.1 ± 0.89 µM respectively, as compared to standard drug ascorbic acid (IC50 = 618 ± 2.0 µM).