Synthesis and biological evaluation of pyrrole-based chalcones as CYP1 enzyme inhibitors,for possible prevention of cancer and overcoming cisplatin resistance

Inhibitors of CYP1 enzymes may play vital roles in the prevention of cancer and overcoming chemo-resistance to anticancer drugs. In this letter, we report synthesis of twenty-three pyrrole based heterocyclic chalcones which were screened for inhibition of CYP1 isoforms. Compound 3n potently inhibited CYP1B1 with an IC₅₀ of ～0.2 μM in Sacchrosomes? and CYP1B1-expressing live human cells. However, compound 3j which inhibited both CYP1A1 and CYP1B1 with an IC₅₀ of ～0.9 µM, using the same systems, also potently antagonized B[a]P-mediated induction of AhR signaling in yeast (IC₅₀, 1.5 µM), fully protected human cells from B[a]P toxicity and completely reversed cisplatin resistance in human cells that overexpress CYP1B1 by restoring cisplatin’s cytotoxicity. Molecular modeling studies were performed to rationalize the observed potency and selectivity of enzyme inhibition by compounds 3j and 3n.     

(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one,a heterocyclic chalcone is a potent and selective CYP1A1 inhibitor and cancer chemopreventive agent

The overexpression of CYP1 family of enzymes is reported to be associated with development of human carcinomas. It has been well reported that CYP1A1 specific inhibitors prevents carcinogenesis. Herein, thirteen pyridine-4-yl series of chalcones were synthesized and screened for inhibition of CYP1 isoforms 1A1, 1B1 and 1A2 in Sacchrosomes? and live human HEK293 cells. The structure-activity relationship analysis indicated that chalcones bearing tri-alkoxy groups (8a and 8k) on non-heterocyclic ring displayed selective inhibition of CYP1A1 enzyme, with IC₅₀ values of 58 and 65?nM, respectively. The 3,4,5-trimethoxy substituted derivative 8a have shown >10-fold selectivity towards CYP1A1 with respect to other enzymes of the CYP1 sub-family and >100-fold selectivity with respect to CYP2 and CYP3 family of enzymes. The potent and selective CYP1A1 inhibitor 8a displayed antagonism of B[a]P mediated activation of aromatic hydrocarbon receptor (AhR) in yeast cells, and also protected human cells from CYP1A1-mediated B[a]P toxicity in human cells. This potent and selective inhibitor of CYP1A1 enzyme have a potential for development as cancer chemopreventive agent.     

Furanoflavones pongapin and lanceolatin B blocks the cell cycle and induce senescence in CYP1A1-overexpressing breast cancer cells

Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is induced (a) by polycyclic aromatic hydrocarbons (PAHs) which can be metabolized by CYP1A1 to carcinogens, and (b) in majority of breast cancers. Hence, phytochemicals or dietary flavonoids, if identified as CYP1A1 inhibitors, may help in preventing PAH-mediated carcinogenesis and breast cancer. Herein, we have investigated the cancer chemopreventive potential of a flavonoid-rich Indian medicinal plant, Pongamia pinnata (L.) Pierre. Methanolic extract of its seeds inhibits CYP1A1 in CYP1A1-overexpressing normal human HEK293 cells, with IC₅₀ of 0.6?µg/mL. Its secondary metabolites, the furanoflavonoids pongapin/lanceolatin B, inhibit CYP1A1 with IC₅₀ of 20?nM. Although the furanochalcone pongamol inhibits CYP1A1 with IC₅₀ of only 4.4?µM, a semisynthetic pyrazole-derivative P5b, has ～10-fold improved potency (IC₅₀, 0.49?μM). Pongapin/lanceolatin B and the methanolic extract of P. pinnata seeds protect CYP1A1-overexpressing HEK293 cells from B[a]P-mediated toxicity. Remarkably, they also block the cell cycle of CYP1A1-overexpressing MCF-7 breast cancer cells, at the G₀-G₁ phase, repress cyclin D1 levels and induce cellular-senescence. Molecular modeling studies demonstrate the interaction pattern of pongapin/lanceolatin B with CYP1A1. The results strongly indicate the potential of methanolic seed-extract and pongapin/lanceolatin B for further development as cancer chemopreventive agents.     

Inhibition of protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase by xanthones from Cratoxylum cochinchinense, and their kinetic characterization

Cratoxylum cochinchinense displayed significant inhibition against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, both of which are key target enzymes to attenuate diabetes and obesity. The compounds responsible for both enzymes inhibition were identified as twelve xanthones (1–12) among which compounds 1 and 2 were found to be new ones. All of them simultaneously inhibited PTP1B with IC₅₀s of (2.4–52.5?µM), and α-glucosidase with IC₅₀ values of (1.7–72.7?µM), respectively. Cratoxanthone A (3) and γ-mangostin (7) were estimated to be most active inhibitors against both PTP1B (IC₅₀?=?2.4?µM for 3, 2.8?µM for 7) and α-glucosidase (IC₅₀?=?4.8?µM for 3, 1.7?µM for 7). In kinetic studies, all isolated xanthones emerged to be mixed inhibitors of α-glucosidase, whereas they behaved as competitive inhibitors of PTP1B. In time dependent experiments, compound 3 showed isomerization inhibitory behavior with following kinetic parameters: K_i^app?=?2.4?µM; k₅?=?0.05001?µM^?1?S^?1 and k₆?=?0.02076?µM^?1?S^?1.     

Discovery of 1,3-diphenyl-1H-pyrazole derivatives containing rhodanine-3-alkanoic acid groups as potential PTP1B inhibitors

Two series of 1,3-diphenyl-1H-pyrazole derivatives containing rhodanine-3-alkanoic acid groups were identified as competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors. Among the compounds studied, IIIv was found to have the best in vitro inhibition activity against PTP1B (IC₅₀?=?0.67?±?0.09?µM) and the best selectivity (9-fold) between PTP1B and T-cell protein tyrosine phosphatase (TCPTP). Molecular docking studies demonstrated that compounds IIIm, IIIv and IVg could occupy simultaneously at both the catalytic site and the adjacent pTyr binding site. These results provide novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.     

Metabolic profiling and biological activities of the aerial parts of Micromeria imbricata Forssk. growing in Saudi Arabia

The hydroalcoholic extract (MIT) of Micromeria imbricata (Forssk.) growing in Saudi Arabia in addition to the chloroform (MIC) and n-butanol (MIB) fractions were investigated for the first time using UPLC-ESI-MS/MS. The analysis revealed the tentative identification of fifty-eight compounds including three organic acids, twenty-five phenolic compounds, three coumarins, two anthocyanins, twenty-one flavonoids, three terpenes, and one miscellaneous. Moreover, the therapeutic potential of M. imbricata (MIT) and its fractions (MIC and MIB) were determined by in vitro evaluation of their cytotoxic, antioxidant, and anti-obesity characteristics. The MIT extract showed the highest phenolic (125.23 ± 0.87 mg gallic acid equivalent/100 gm extract) and flavonoid (112.24 ± 2.45 mg quercetin equivalent/100 gm extract) contents followed by n-butanol and chloroform fractions. The MIT extract revealed a potent cytotoxic activity against HepG-2 (Hepatocellular carcinoma) and MCF-7 (Breast carcinoma) with IC₅₀ 28.5 ± 2.0 and 35.2 ± 1.2 µg/mL, respectively. Additionally, the tested hydroalcoholic extract exhibited a significant DPPH scavenging activity with SC₅₀ 28.4 ± 1.2 µg/mL and a remarkable lipase inhibitory activity with IC₅₀ 54.2 ± 1.2 µg/mL. In conclusion, the current study presents the first insights into the phytochemical constituents and pharmacological properties of M. imbricata extract and its chloroform and n-butanol fractions. The results revealed that M. imbricata hydroalcoholic extract might be a prolific source of bioactive constituents with potent antioxidant, cytotoxic and anti-obesity potential. It might be a natural alternative therapy and nutritional strategy for obesity treatment.     

Antioxidant and angiotensin-converting enzyme (ACE) inhibitory activity of thymosin alpha-1 (Thα1) peptide

In this research, the antioxidant property of thymosin alpha-1 (Thα1) peptide was investigated through various antioxidant methods. Thα1 showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (IC₅₀ = 20 µM) and its 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenging reached 45.33% at 80 µM (IC₅₀ = 85 µM). In addition, hydroxyl and superoxide radical scavenging of Thα1 peptide exhibited a concentration-depended manner. The IC₅₀ values of hydroxyl and superoxide radical scavenging were estimated to be 82 µM and 20 µM, respectively. The effect of Thα1 on eliminating superoxide radicals was higher (62.23%) than other antioxidant assays. Moreover, the antioxidant activity of Thα1 peptide was evaluated by measuring cellular reactive oxygen species (ROS). Results indicated that Thα1 decreased the generation of ROS level in 1321 N1 human neural asterocytoma cells. The inhibitory effect of Thα1 on angiotensin-converting enzyme (ACE) was determined. The kinetic parameters (K_m and V_max) and the inhibition pattern were examined. Based on the Lineweaver-Burk plot, Thα1 displayed a mixed inhibition pattern. The IC₅₀ and K_i values of Thα1 were 0.8 µM and 3.33 µM, respectively. Molecular modeling suggested that Thα1 binds to ACE-domains with higher affinity binding to N-domain with the binding energy of −22.87 kcal/mol. Molecular docking indicated that Thα1 interacted with ACE enzyme (N- and C-domains) due to electrostatic, hydrophobic, and hydrogen forces. Our findings suggested that Thα1 possess a multifunctional peptide with dual antioxidant and ACE-inhibitory properties. Further researches are needed to investigate the antioxidant and anti-hypertensive effect of Thα1 both in vitro and in vivo.     

Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana

Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC₅₀ values 3.49?±?0.02, 4.17?±?0.03 and 87.52?±?0.03?µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds.     

Structure optimization and bioactivity evaluation of ThDP analogs targeting cyanobacterial pyruvate dehydrogenase E1

Harmful cyanobacteria bloom (HCB) has occurred frequently in recent years and it is urgent to develop novel algicides to deal with this problem. In this paper, a series of novel thiamin diphosphate (ThDP) analogs 5a?5g were designed and synthesized targeting cyanobacterial pyruvate dehydrogenase complex E1 (Cy-PDHc E1). Our results showed that compounds 5a?5g have higher inhibitory activities against Cy-PDHc E1 (IC₅₀ 9.56–3.48 µM) and higher inhibitory activities against two model cyanobacteria strains Synechocystis sp PCC6803 (EC₅₀ 2.03–1.58 µM) and Microcystis aeruginosa FACHB905 (EC₅₀ 1.86–0.95 µM). Especially, compound 5b displayed highest inhibitory activities (IC₅₀ = 3.48 µM) against Cy-PDHc E1 and powerful inhibitory activities against cyanobacteria Synechocystis sp PCC6803 (EC₅₀ = 1.58 µM) and Microcystis aeruginosa FACHB905 (EC₅₀ = 1.04 µM). Moreover, the inhibitory activities of compound 5b were even higher than those of copper sulfate (EC₅₀ = 2.02 and 1.71 µM separately) which has been widely used as algicide against cyanobacteria PCC6803 and FACHB905. The more important was that compound 5b display much higher inhibitory selectivity between Cy-PDHc E1 (Inhibitory rate 97.4%) and porcine PDHc E1 (Inhibitory rate 11.8%) under the same concentration (100 μM). The inhibition kinetic experiment and molecular docking research showed that compound 5b can inhibit Cy-PDHc E1 by occupying the ThDP-binding pocket and then blocking Cy-PDHc E1 bound to ThDP as competitive inhibitor. The imagines of SEM and TEM showed that cellular microstructures were heavily destroyed under compound 5b stress. Our results demonstrated compound 5b could be taken as a potential lead compound targeting Cy-PDHc E1 to obtain environment-friendly algicide for harmful cyanobacterial blooms control.     

Design,synthesis and biological evaluation of some tetrazole acetamide derivatives as novel non-carboxylic PTP1B inhibitors

A series of ten N-(3-(1H-tetrazole-5-yl)phenyl)acetamide derivatives (NM-07 to NM-16) designed from a lead molecule identified previously in our laboratory were synthesized and evaluated for protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. Among the synthesized molecules, NM-14, a 5-Cl substituted benzothiazole analogue elicited significant PTP1B inhibition with an IC₅₀ of 1.88 µM against reference standard suramin (IC₅₀ ≥ 10 µM). Furthermore, this molecule also showed good in vivo antidiabetic activity which was comparable to that of standard antidiabetic drugs metformin and glimepiride. Overall, the results of the study clearly reveal that the reported tetrazole derivatives especially NM-14 are valuable prototypes for the development of novel non-carboxylic inhibitors of PTP1B with antidiabetic potential.     

Cytochrome P450 binding studies of novel tacrine derivatives: Predicting the risk of hepatotoxicity

The 1,2,3,4-tetrahydroacridine derivative tacrine was the first drug approved to treat Alzheimer’s disease (AD). It is known to act as a potent cholinesterase inhibitor. However, tacrine was removed from the market due to its hepatotoxicity concerns as it undergoes metabolism to toxic quinonemethide species through the cytochrome P450 enzyme CYP1A2. Despite these challenges, tacrine serves as a useful template in the development of novel multi-targeting anti-AD agents. In this regard, we sought to evaluate the risk of hepatotoxicity in a series of C9 substituted tacrine derivatives that exhibit cholinesterase inhibition properties. The hepatotoxic potential of tacrine derivatives was evaluated using recombinant cytochrome (CYP) P450 CYP1A2 and CYP3A4 enzymes. Molecular docking studies were conducted to predict their binding modes and potential risk of forming hepatotoxic metabolites. Tacrine derivatives compound 1 (N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) and 2 (6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) which possess a C9 3,4-dimethoxybenzylamino substituent exhibited weak binding to CYP1A2 enzyme (1, IC₅₀ = 33.0 µM; 2, IC₅₀ = 8.5 µM) compared to tacrine (CYP1A2 IC₅₀ = 1.5 µM). Modeling studies show that the presence of a bulky 3,4-dimethoxybenzylamino C9 substituent prevents the orientation of the 1,2,3,4-tetrahydroacridine ring close to the heme-iron center of CYP1A2 thereby reducing the risk of forming hepatotoxic species.     

Investigation of cytochrome P450 inhibitory properties of maslinic acid,a bioactive compound from Olea europaea L., and its structure–activity relationship

Maslinic acid (MA), the main pentacyclic triterpene of Olea europaea L. fruit, possesses a variety of pharmacological actions, including hypoglycemic, antioxidant, cardioprotective and antitumoral activities. Despite its importance, little is known about its effects on the cytochrome P450 (CYP) activity in both humans and animals. Therefore, the aim of this study was to investigate the effects of MA on the CYP 1A2, 2C9/11, 2D1/6, 2E1 and 3A2/4 activities by human and rat liver microsomes and specific CYP isoforms. In humans, MA only weakly inhibited CYP3A4 activity in human liver microsomes and specific CYP3A4 isoform with IC₅₀ value at 46.1 and 62.3 µM, respectively. In rats, MA also exhibited weak inhibition on CYP2C11, CYP2E1 and CYP3A2 activities with IC₅₀ values more than 100 µM. Enzyme kinetic studies showed that the MA was not only a competitive inhibitor of CYP3A4 in humans, but also a competitive inhibitor of CYP2C11 and 3A2 in rats, with K_i of 18.4, 98.7 and 66.3 µM, respectively. Moreover, the presence of hydroxyl group at C-2 position of triterpenic acid in MA compared with oleanolic acid could magnify its competitive inhibition on human CYP3A4 activity. The relatively high K_i values of MA would have a low potential to cause the possible toxicity and drug interactions involving CYP enzymes, thus suggesting a sufficient safety for its putative use as a nutraceutical taken together with drugs.     

Design,synthesis, SAR and biological investigation of 3-(carboxymethyl)rhodanine and aminothiazole inhibitors of Mycobacterium tuberculosis Zmp1

Sixteen 3-(carboxymethyl)rhodanines, and twelve aminothiazoles as rhodanine-mimetics were designed, synthesized and tested as inhibitors of the Zmp1 enzyme from Mycobacterium tuberculosis (Mtb). Almost all rhodanines (5a–d, 5f–n, and 7a–b) exhibited Zmp1 inhibition with IC₅₀ values in the range 1.3–43.9?µM, whereas only aminothiazoles 12b and 12d proved active with IC₅₀ values of 41.3 and 35.7?µM, respectively. Structure-activity relationships (SAR) were coupled with molecular modeling studies to highlight structural determinants for Zmp1 inhibition. Moreover, rhodanines 5a and 5c induced 23.4 and 53.8% of Mtb growth inhibition in THP-1 infected cells, respectively, at the non-toxic concentration of 10?µg/ml. This work represents a step forward in targeting Zmp1 by small molecules.     

In vitro effects of some flavonoids and phenolic acids on human pyruvate kinase isoenzyme M2

Pyruvate kinase isoenzyme M2 (PKM2) is one of the most important control point enzyme in glycolysis pathway. Hence, its inhibitors and activators are currently considered as the potential anticancer agents. The effect of 28 polyphenolic compounds on the enzyme activity was investigated in vitro. Among these compounds, neoeriocitrin, (?)-catechin gallate, fisetin, (±)-taxifolin and (?)-epicatechin have the highest inhibition effect with IC₅₀ value within 0.65–1.33?µM range. Myricetin and quercetin 3-β-d-glucoside exhibited the highest activation effect with 0.51 and 1.34?µM AC₅₀ values, respectively. Twelve of the compounds showed inhibition effect within 7–38?µM range of IC₅₀ value. Sinapinic acid and p-coumaric acid showed an activation effect with 26.2 and 22.2?µM AC₅₀ values, respectively. The results propose that the polyphenolics may be the potential PKM2 inhibitors/activators, and they may be used as lead compounds for the synthesis of new inhibitors or activators of this enzyme.     

A multicomponent approach in the discovery of indoleamine 2,3-dioxygenase 1 inhibitors: Synthesis,biological investigation and docking studies

Indoleamine 2,3-dioxygenase plays a crucial role in immune tolerance and has emerged as an attractive target for cancer immunotherapy. In this study, the Passerini and Ugi multicomponent reactions have been employed to assemble a small library of imidazothiazoles that target IDO1. While the p-bromophenyl and the imidazothiazole moieties have been kept fixed, a full SAR study has been performed on the side-chain, leading to the discovery of nine compounds with sub-micromolar IC₅₀ values in the enzyme-based assay. Compound 7d, displaying a α-acyloxyamide substructure, is the most potent compound, with an IC₅₀ value of 0.20?µM, but a low activity in a cell-based assay. Compound 6o, containing a α-acylaminoamide moiety, shows an IC₅₀ value of 0.81?µM in the IDO1-based assay, a full biocompatibility at 10?µM, together with a modest inhibitory activity in A375 cells. Molecular docking studies show that both 7d and 6o display a unique binding mode in the IDO1 active site, with the side-chain protruding in an additional pocket C, where a crucial hydrogen bond is formed with Lys238. Overall, this work describes an isocyanide based-multicomponent approach as a straightforward and versatile tool to rapidly access IDO1 inhibitors, providing a new direction for their future design and development.     

In vitro effect of important herbal active constituents on human cytochrome P450 1A2 (CYP1A2) activity

This study was designed to investigate eight herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid) on their potential inhibitory effects on human cytochrome P450 1A2 (CYP1A2) activity. A fluorescence-based enzyme assay was performed by co-incubating human cDNA-expressed CYP1A2 with its selective probe substrate, 3-cyano-7-ethoxycoumarin (CEC), in the absence or presence of various concentrations of herbal active constituents. The metabolite (cyano-hydroxycoumarin) formed was subsequently measured in order to obtain IC₅₀ values. The results indicated that only eupatorin and sinensetin moderately inhibited CYP1A2 with IC₅₀ values of 50.8 and 40.2 μM, while the other active compounds did not significantly affect CYP1A2 activity with IC₅₀ values more than 100 μM. K_i values further determined for eupatorin and sinensetin were 46.4 and 35.2 μM, respectively. Our data indicated that most of the investigated herbal constituents have negligible CYP1A2 inhibitory effect. In vivo studies however may be warranted to ascertain the inhibitory effect of eupatorin and sinensetin on CYP1A2 activity in clinical situations     

Synthesis and antitumor activity of novel 2, 3-didithiocarbamate substituted naphthoquinones as inhibitors of pyruvate kinase M2 isoform

The M2 isoform of pyruvate kinase (PKM2) is a potential antitumor therapeutic target. In this study, we designed and synthesised a series of 2, 3-didithiocarbamate substituted naphthoquinones as PKM2 inhibitors based on the lead compound 3k that we previously reported. Among them, compound 3f (IC₅₀?=?1.05?±?0.17 µM) and 3h (IC₅₀?=?0.96?±?0.18 µM) exhibited potent inhibition of PKM2, and their inhibitory activities are superior to compound 3k (IC₅₀?=?2.95?±?0.53 µM) and the known PKM2 inhibitor shikonin (IC₅₀?=?8.82?±?2.62 µM). In addition, we evaluated in vitro antiproliferative effects of target compounds using MTS assay. Most target compounds exhibited dose-dependent cytotoxicity with IC₅₀ values in nanomolar concentrations against HCT116, MCF7, Hela, H1299 and B16 cells. These small molecule PKM2 inhibitors not only provide candidate compounds for cancer therapy, but also offer a tool to probe the biological effects of PKM2 inhibition on cancer cells.     

Synthesis and characterization of 5,7-dihydroxyflavanone derivatives as novel protein tyrosine phosphatase 1B inhibitors

A series of 5,7-dihydroxyflavanone derivatives were synthesized and identified as reversible and competitive protein tyrosine phosphatase (PTP) 1B inhibitors with IC₅₀ values in the micromolar range. Compound 4k had the most potent in vitro inhibition activity against PTP1B (IC₅₀ = 2.37?±?0.37 μM) and the greatest selectivity (3.7-fold) for PTP1B relative to T-cell protein tyrosine phosphatase. Cell-based studies revealed that 4k was membrane-permeable and enhanced insulin receptor tyrosine phosphorylation in CHO/hIR cells.     

N-(Pyridin-3-yl)benzamides as selective inhibitors of human aldosterone synthase (CYP11B2)

A series of 23 N-(Pyridin-3-yl)benzamides was synthesized and evaluated for their potential to inhibit human steroid-11β-hydroxylase (CYP11B1) and human aldosterone synthase (CYP11B2). The most potent and selective CYP11B2 inhibitors (IC₅₀ values 53-166 nM) were further evaluated for their potential to inhibit human CYP17 and CYP19, and no inhibition was observed. Clear evidence was shown for N-(Pyridin-3-yl)benzamides to be a highly selective class of CYP11B2 inhibitors in vitro.