Evaluation of oxindole derivatives as a potential anticancer agent against breast carcinoma cells: In vitro, in silico, and molecular docking study.

In this study we have performed the in vitro anticancer activity of spiro oxindole derivatives against MCF-7 (human Adreno carcinoma) and MDA-MB-231 (triple negative breast cancer) cell lines to propose a possible role of these derivatives in the treatment of cancer. Compound 6, which has an N-benzyl substitution with a chloro group on the indolin-2-one scaffold, had the most potent activity against MCF-7 (3.55 ± 0.49 µM) and MDA-MB-231 (4.40 ± 0.468 µM) of all the synthesized molecules. A normal mouse embryonic fibroblast (NIH/3 T3) cell line was used to test the cellular toxicity of these derivatives. The results showed that none of the compounds were cytotoxic to normal cells. In addition, pharmacokinetic (ADME) and toxicity study profiles were predicted in silico. All the synthesized derivatives (1 to 7) demonstrated the necessary physicochemical properties for bioavailability. Finally, in vitro results of promising compound 6 were validated using molecular docking and dynamic simulation studies, which revealed their binding affinities and conformational stability in the binding cavity. Thus, these derivatives may serve as lead structures for a new generation of anticancer agents.

Indole Derivatives as New Structural Class of Potent and Antiproliferative Inhibitors of Monocarboxylate Transporter 1 (MCT1; SLC16A1).

The solute carrier (SLC) monocarboxylate transporter 1 (MCT1; SLC16A1) represents a promising target for the treatment of cancer; however, the MCT1 modulator landscape is underexplored with only roughly 100 reported compounds. To expand the knowledge about MCT1 modulation, we synthesized a library of 16 indole-based molecules and subjected these to a comprehensive biological assessment platform. All compounds showed functional inhibitory activities against MCT1 at low nanomolar concentrations and great antiproliferative activities against the MCT1-expressing cancer cell lines A-549 and MCF-7, while the compounds were selective over MCT4 (SLC16A4). Lead compound 24 demonstrated a greater potency than the reference compound, and molecular docking revealed strong binding affinities to MCT1. Compound 24 led to cancer cell cycle arrest as well as apoptosis, and it showed to sensitize these cancer cells toward an antineoplastic agent. Strikingly, compound 24 had also significant inhibitory power against the multidrug transporter ABCB1 and showed to reverse ABCB1-mediated multidrug resistance (MDR).

Applications of Nanotechnology-based Approaches to Overcome Multi-drug Resistance in Cancer.

Cancer is one of the leading causes of death worldwide. Chemotherapy and radiation therapy are the major treatments used for the management of cancer. Multidrug resistance (MDR) is a major hindrance faced in the treatment of cancer and is also responsible for cancer relapse. To date, several studies have been carried out on strategies to overcome or reverse MDR in cancer. Unfortunately, the MDR reversing agents have been proven to have minimal clinical benefits, and eventually, no improvement has been made in therapeutic efficacy to date. Thus, several investigational studies have also focused on overcoming drug resistance rather than reversing the MDR. In this review, we focus primarily on nanoformulations regarded as a novel approach to overcome or bypass the MDR in cancer. The nanoformulation systems serve as an attractive strategy as these nanosized materials selectively get accumulated in tumor tissues, thereby improving the clinical outcomes of patients suffering from MDR cancer. In the current work, we present an overview of recent trends in the application of various nano-formulations, belonging to different mechanistic classes and functionalization like carbon nanotubes, carbon nanohorns, carbon nanospheres, liposomes, dendrimers, etc., to overcome MDR in cancer. A detailed overview of these techniques will help researchers in exploring the applicability of nanotechnologybased approaches to treat MDR.

Monocarboxylate transporter 1 and 4 inhibitors as potential therapeutics for treating solid tumours: A review with structure-activity relationship insights.

Development of multidrug resistance (MDR) is one of the major causes leading to failure of cancer chemotherapy and radiotherapy. Monocarboxylate transporters (MCTs) MCT1 and MCT4, which are overexpressed in solid tumours, play a very important role in cancer cell survival and proliferation. These lactate transporters work complimentarily to drive lactate shuttle in tumour cells, which results in maintenance of H+ ion (pH) balance necessary for their survival. Inhibition of these transmembrane proteins has been demonstrated as a novel strategy to treat drug resistant solid cancers. Presently, only a few small molecule MCT1 inhibitors such as AZD3965 and AR-C155858 are known with clinical potential. Even lesser mention of MCT4 inhibitors, which include molecules having scaffolds such as pyrazole and indazole, is available in the literature. Current overview presents the status of recent developments undertaken in identification of efficacious MCT1 and/or MCT4 inhibitors as a potential anticancer therapy overcoming MDR. Further, detailed structure-activity relationships for different classes of compounds has been proposed to streamline the understandings learnt from ongoing research work. Through this review, we aim to highlight the importance of these excellent targets and facilitate future development of selective, potent and safe MCT1 and/or MCT4 inhibitors as promising chemotherapy for drug resistant cancer.

Inhibitors of inosine 5'-monophosphate dehydrogenase as emerging new generation antimicrobial agents.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a vital enzyme involved in the de novo synthesis of guanine nucleotides. IMPDH catalyzes a crucial step of converting IMP into XMP that is further converted into GMP. Microbial infections rely on the rapid proliferation of bacteria, and this requires the rate-limiting enzyme IMPDH to expand the guanine nucleotide pool and hence, IMPDH has recently received lots of attention as a potential target for treating infections. Owing to the structural and kinetic differences in the host IMPDH and bacterial IMPDH, a selective targeting is possible and is a crucial feature in the development of new potent and selective inhibitors of bacterial IMPDH. Earlier screening of small molecules revealed a structural requirement for the bacterial/protozoal IMPDH. Early optimization of benzimidazole and benzoxazole scaffolds led to the discovery of new potent and selective inhibitors of pathogenic IMPDH. Further research is vastly focused on the development of highly potent and selective inhibitors of various bacterial IMPDHs. Such studies reveal the importance of this excellent target for treating infectious diseases. The current review focuses on the recent developments in the discovery and development of selective inhibitors of bacterial/protozoal IMPDH with emphasis on the inhibition mechanism and structure-activity relationship.

Cancer cell fusion: a potential target to tackle drug-resistant and metastatic cancer cells.

Cell fusion is an integral, established phenomenon underlying various physiological processes in the cell cycle. Although research in cancer metastasis has hypothesised numerous molecular mechanisms and signalling pathways responsible for invasion and metastasis, the origin and progression of metastatic cells within primary tumours remains unclear. Recently, the role of cancer cell fusion in cancer metastasis and development of multidrug resistance (MDR) in tumours has gained prominence. However, evidence remains lacking to justify the role of cell fusion in cancer metastasis and drug resistance. Here, we highlight plausible mechanisms governing cell fusion with different cell types in the tumour microenvironment (TME), the clinical relevance of cancer cell fusion, its potential as a target for overcoming MDR and inhibiting metastasis, and putative modes of treatment.

Design, synthesis and biological evaluation of Helicobacter pylori inosine 5'-monophosphate dehydrogenase (HpIMPDH) inhibitors. Further optimization of selectivity towards HpIMPDH over human IMPDH2.

Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes a crucial step in guanine nucleotide biosynthesis, thereby governing cell proliferation. In contrast to mammalian IMPDHs, microbial IMPDHs are relatively less explored as potential targets for antimicrobial drug discovery. In continuation with our previous work, here we report the discovery of moderately potent and highly selective Helicobacter pylori IMPDH (HpIMPDH) inhibitors. The present study is mainly focused around our previously identified, modestly potent and relatively nonselective (for HpIMPDH over human IMPDH2) hit molecule IX (16i). In an attempt to optimize the selectivity for the bacterial enzyme, we screened a set of 48 redesigned new chemical entities (NCEs) belonging to 5-aminoisobenzofuran-1(3H)-one series for their in vitro HpIMPDH and human IMPDH2 inhibition. A total of 12 compounds (hits) demonstrated ≥70% HpIMPDH inhibition at 10 µM concentration; none of the hits were active against hIMPDH2. Compound 24 was found to be the most potent and selective molecule (HpIMPDH IC50 = 2.21 µM) in the series. The study reaffirmed the utility of 5-aminoisobenzofuran-1(3H)-one as a promising scaffold with great potential for further development of potent and selective HpIMPDH inhibitors.

Synthesis and In Vitro Enzymatic Studies of New 3-Aryldiazenyl Indoles as Promising Helicobacter pylori IMPDH Inhibitors.

BACKGROUND & OBJECTIVE: Helicobacter pylori infection is one of the primary causes of peptic ulcer followed by gastric cancer in the world population. Due to increased occurrences of multi-drug resistance to the currently available antibiotics, there is an urgent need for a new class of drugs against H. pylori. Inosine 5'-monophosphate dehydrogenase (IMPDH), a metabolic enzyme plays a significant role in cell proliferation and cell growth. It catalyses guanine nucleotide synthesis. IMPDH enzyme has been exploited as a target for antiviral, anticancer and immunosuppressive drugs. Recently, bacterial IMPDH has been studied as a potential target for treating bacterial infections. Differences in the structural and kinetic parameters of the eukaryotic and prokaryotic IMPDH make it possible to target bacterial enzyme selectively. METHODS: In the current work, we have synthesised and studied the effect of substituted 3-aryldiazenyl indoles on Helicobacter pylori IMPDH (HpIMPDH) activity. The synthesised molecules were examined for their inhibitory potential against recombinant HpIMPDH. RESULTS: In this study, compounds 1 and 2 were found to be the most potent inhibitors amongst the database with IC50 of 0.8 ± 0.02µM and 1 ± 0.03 µM, respectively. CONCLUSION: When compared to the most potent known HpIMPDH inhibitor molecule C91, 1 was only four-fold less potent and can be a good lead for further development of selective and potent inhibitors of HpIMPDH.

Identification of selective inhibitors of Helicobacter pylori IMPDH as a targeted therapy for the infection.

Helicobacter pylori (H. pylori), the major cause of several gastric disorders has been recognied as a type I carcinogen. By virtue of resistance developed by H. pylori strains, currently used antibiotic based treatments rather demonstrate high failure rates. Hence, there is an emerging need for identification of new targets to treat H. pylori infection. Inosine-5'-monophosphate dehydrogenase (IMPDH) has been studied as a potential target to treat H. pylori infection. Here, a detailed enzyme kinetic study of recombinant expressed H. pylori inosine-5'-monophosphate dehydrogenase (HpIMPDH) is presented. A new in-house synthesized indole-based scaffold is identified as an inhibitor for HpIMPDH. These indole-based compounds showed non-competitive inhibition against IMP and NAD+ whereas the benzimidazole compounds were found be uncompetitive inhibitors. The new indole scaffold ensures specificity due to its high selectivity for bacterial IMPDH over human IMPDH II. Our work aims to overcome the drawback of existing inhibitors by introducing new indole scaffold for targeting bacterial IMPDH.

Novel chalcone and flavone derivatives as selective and dual inhibitors of the transport proteins ABCB1 and ABCG2.

During cancer chemotherapy, certain cancers may become cross-resistant to structurally diverse antineoplastic agents. This so-called multidrug resistance (MDR) is highly associated with the overexpression of ATP-binding cassette (ABC) transport proteins. These membrane-bound efflux pumps export a broad range of structurally diverse endo- and xenobiotics, including chemically unrelated anticancer agents. This translocation of drugs from the inside to the outside of cancer cells is mediated at the expense of ATP. In the last 40 years, three ABC transporters - ABCB1 (P-gp), ABCC1 (MRP1), and ABCG2 (BCRP) - have mainly been attributed to the occurrence of MDR in cancer cells. One of the strategies to overcome MDR is to inhibit the efflux transporter function by small-molecule inhibitors. In this work, we investigated new chalcone- and flavone-based compounds for selective as well as broad-spectrum inhibition of the stated transport proteins. These include substituted chalcones with variations at rings A and B, and flavones with acetamido linker at position 3. The synthesized molecules were evaluated for their inhibitory potential against ABCB1, ABCC1, and ABCG2 in calcein AM and pheophorbide A assays. In further investigations with the most promising candidates from each class, we proved that ABCB1- and ABCG2-mediated MDR could be reversed by the compounds. Moreover, their intrinsic toxicity was found to be negligible in most cases. Altogether, our findings contribute to the understanding of ABC transport proteins and reveal new compounds for ongoing evaluation in the field of ABC transporter-mediated MDR.

Water-mediated intermolecular interactions in 1,2-O-cyclohexylidene-myo-inositol: a quantitative analysis.

The syntheses of new myo-inositol derivatives have received much attention due to their important biological activities. 1,2-O-Cyclohexylidene-myo-inositol is an important intermediate formed during the syntheses of certain myo-inositol derivatives. We report herein the crystal structure of 1,2-O-cyclohexylidene-myo-inositol dihydrate, C12H20O6·2H2O, which is an intermediate formed during the syntheses of myo-inositol phosphate derivatives, to demonstrate the participation of water molecules and hydroxy groups in the formation of several intermolecular O-H...O interactions, and to determine a low-energy conformation. The title myo-inositol derivative crystallizes with two water molecules in the asymmetric unit in the space group C2/c, with Z = 8. The water molecules facilitate the formation of an extensive O-H...O hydrogen-bonding network that assists in the formation of a dense crystal packing. Furthermore, geometrical optimization and frequency analysis was carried out using density functional theory (DFT) calculations with B3LYP hybrid functionals and 6-31G(d), 6-31G(d,p) and 6-311G(d,p) basis sets. The theoretical and experimental structures were found to be very similar, with only slight deviations. The intermolecular interactions were quantitatively analysed using Hirshfeld surface analysis and 2D (two-dimensional) fingerplot plots, and the total lattice energy was calculated.

The combination of quinazoline and chalcone moieties leads to novel potent heterodimeric modulators of breast cancer resistance protein (BCRP/ABCG2).

During the last decade it has been found that chalcones and quinazolines are promising inhibitors of ABCG2. The combination of these two scaffolds offers a new class of heterocyclic compounds with potentially high inhibitory activity against ABCG2. For this purpose we investigated 22 different heterodimeric derivatives. In this series only methoxy groups were used as substituents as these had been proven superior for inhibitory activity of chalcones. All compounds were tested for their inhibitory activity, specificity and cytotoxicity. The most potent ABCG2 inhibitor in this series showed an IC50 value of 0.19 µM. It possesses low cytotoxicity (GI50 = 93 µM), the ability to reverse MDR and is nearly selective toward ABCG2. Most compounds containing dimethoxy groups showed slight activity against ABCB1 too. Among these three compounds (17, 19 and 24) showed even higher activity toward ABCB1 than ABCG2. All inhibitors were further screened for their effect on basal ATPase activity. Although the basal ATPase activity was partially stimulated, the compounds were not transported by ABCG2. Thus, quinazoline-chalcones are a new class of effective ABCG2 inhibitors.

Design of inhibitors of BCRP/ABCG2.

BCRP/ABCG2, a second member of ABC transporter subclass G, has been shown to be overexpressed in several solid tumors, acute myelogenous leukemia and chronic myeloid leukemia. A variety of chemically unrelated anticancer drugs have been found to be transported by ABCG2 leading to their lower intracellular accumulation and hence causing chemoresistance. Until now several efforts have been taken to identify potent and selective inhibitors of ABCG2. Recent studies carried out to deign BCRP inhibitors have been able to point out the effect of the substitution pattern in compound scaffolds on the potency, selectivity and cytotoxicity of ABCG2 inhibitors.

Evaluation of dual P-gp-BCRP inhibitors as nanoparticle formulation.

Overcoming multidrug resistance (MDR) in cancer is a major challenge and efforts are on-going to develop inhibitors against the most characterized and ubiquitous MDR transporters: P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1) and breast cancer resistance protein (BCRP). Recently reported, two 4-anilinoquinazolines (compounds 1 and 2), demonstrate potential MDR reversal activity against BCRP and to a lesser extent, P-gp. In this work, we formulated the compounds as polymeric nanoparticles (NPs) and assessed their MDR inhibitory activity in relevant BCRP and P-gp over-expressing cell line models. Particles in the size range 300-365nm with a loading efficiency of 69% (compound 1 NP) and 77% (compound 2 NP) respectively were obtained. BCRP inhibition was observed in Hoechst 33342 and pheophorbide A assays while P-gp inhibition was evaluated in calcein AM and rhodamine-123 assays. In cytotoxicity studies, while BCRP expressing cells showed complete reversal of drug resistance in nearly all treatment groups (both compounds and their respective NP); a higher reversal in NP treated group was obtained as compared with inhibitory compound treated group in P-gp expressing cells. These results demonstrate promising inhibitory activity of both formulations, especially against P-gp expressing cells; which is possibly due to a prolonged presence of encapsulated compounds in NPs and consequently a prolonged sensitization of transmembrane drug transporter. These formulations can therefore be considered as dual-transporter inhibitors and it is imperative to investigate both inhibitors in animal models of MDR owing to the presence of multiple efflux transporters in several cancer models.

Characterization of 3-methoxy flavones for their interaction with ABCG2 as suggested by ATPase activity.

Breast Cancer Resistance Protein (BCRP/ABCG2) belongs to the superfamily of ATP binding cassette (ABC) transporters. Characteristic of some of these transporter proteins is the transport of a variety of structurally unrelated substances against a concentration gradient by using the energy of ATP hydrolysis. ABCG2 has been found to confer multidrug resistance (MDR) in cancer cells. Several anticancer drugs have been identified as ABCG2 substrates including mitoxantrone, etoposide and topotecan. As inhibition of the transporter is one of the strategies to overcome MDR, we have synthesized and tested several 3-methoxy flavones and investigated them for their ABCG2 inhibition. Among these, pentamethyl quercetin (compound 4) and pentamethyl morin (compound 5) were found to be fluorescent and hence screened for their possible transport by ABCG2 using confocal microscopy. This study showed that pentamethyl quercetin was far less accumulated in ABCG2 overexpressing MDCK BCRP cells as compared to MDCK sensitive cells, suggesting possible efflux of this compound by ABCG2. Pentamethyl morin showed no visible difference in both cell lines. Based on this observation, we studied several other fluorescent 3-methoxy flavones for their accumulation in ABCG2 overexpressing cells. To confirm the substrate or inhibitor nature of the tested compounds, these compounds were further investigated by ATPase assay. If stimulation of the transporter ATPase activity is detected, one can conclude that the compound is probably a transported substrate. All compounds except pentamethyl morin (compound 5) and tetramethyl quercetin (compound 6) were found to stimulate ATPase activity pointing to possible substrates despite being potent inhibitors of ABCG2.

Investigation of quinazolines as inhibitors of breast cancer resistance protein (ABCG2).

Chemotherapy is one of the major forms of cancer treatment. Unfortunately, tumors are prone to multidrug resistance leading to failure of treatment. Breast cancer resistance protein (BCRP), the second member of ABC transporter subfamily G, has been found to play a major role in drug efflux and hence multidrug resistance. Until now, very few potent and selective BCRP inhibitors like Ko143 have been identified. In the search for more potent and selective BCRP inhibitors, we synthesized and investigated a series of differently substituted quinazoline compounds. Several variations at positions 2, 4, 6 and 7 of the quinazoline scaffold were carried out to develop a structure-activity-relationship analysis for these compounds. It was found that compounds bearing a phenyl substituent at position 2 of the 4-anilinoquinazoline scaffold were most potent. On the aniline ring at position 4 of the quinazoline moiety substituents like NO2, CN, CF3 led to very high BCRP inhibition potencies. The most potent compounds were further investigated for their intrinsic cytotoxicity and their ability to reverse the multidrug resistance. Compound 20, an anilinoquinazoline bearing a phenyl ring at position 2 and meta-nitro substitution on the 4-anilino ring, was found to have the highest therapeutic ratio. The most active compounds from each variation were also investigated for their effect on BCRP expression. It was found that compound 20 has no significant effect on BCRP expression, while compound 31 decreased the surface BCRP expression. The only difference in the two compounds was the presence of a 3,4-dimethoxyphenyl ring in compound 31 instead of phenyl substitution at position 2 of the quinazoline moiety. From the study of all target compounds, compound 20 was the most prominent compound having inhibitory potency even higher than Ko143, the most potent BCRP inhibitor known. Compound 20 was also found to be selective towards BCRP with a very high therapeutic ratio.

Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2.

Multidrug resistance (MDR) often leads to a failure of cancer chemotherapy. Breast Cancer Resistance Protein (BCRP/ABCG2), a member of the superfamily of ATP binding cassette proteins has been found to confer MDR in cancer cells by transporting molecules with amphiphilic character out of the cells using energy from ATP hydrolysis. Inhibiting BCRP can be a solution to overcome MDR. We synthesized a series of flavones, 7,8-benzoflavones and 5,6-benzoflavones with varying substituents at positions 3, 3' and 4' of the (benzo)flavone structure. All synthesized compounds were tested for BCRP inhibition in Hoechst 33342 and pheophorbide A accumulation assays using MDCK cells expressing BCRP. All the compounds were further screened for their P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP1) inhibitory activity by calcein AM accumulation assay to check the selectivity towards BCRP. In addition most active compounds were investigated for their cytotoxicity. It was observed that in most cases 7,8-benzoflavones are more potent in comparison to the 5,6-benzoflavones. In general it was found that presence of a 3-OCH3 substituent leads to increase in activity in comparison to presence of OH or no substitution at position 3. Also, it was found that presence of 3',4'-OCH3 on phenyl ring lead to increase in activity as compared to other substituents. Compound 24, a 7,8-benzoflavone derivative was found to be most potent being 50 times selective for BCRP and showing very low cytotoxicity at higher concentrations.

4-Substituted-2-phenylquinazolines as inhibitors of BCRP.

We investigated several 2-phenylquinazolines with different substitutions at position 4 for their BCRP inhibition. Compounds with phenyl ring attached via an amine-containing linker at position 4 were found to be potent inhibitors of BCRP. In general compounds with meta substitution of phenyl ring at position 4 were found to have higher inhibitory effect, compound 12 being the most potent and selective towards BCRP.

Investigation of chalcones and benzochalcones as inhibitors of breast cancer resistance protein.

Breast cancer resistance protein (BCRP/ABCG2) belongs to the ATP binding cassette family of transport proteins. BCRP has been found to confer multidrug resistance in cancer cells. A strategy to overcome resistance due to BCRP overexpression is the investigation of potent and specific BCRP inhibitors. The aim of the current study was to investigate different multi-substituted chalcones for their BCRP inhibition. We synthesized chalcones and benzochalcones with different substituents (viz. OH, OCH(3), Cl) on ring A and B of the chalcone structure. All synthesized compounds were tested by Hoechst 33342 accumulation assay to determine inhibitory activity in MCF-7 MX and MDCK cells expressing BCRP. The compounds were also screened for their P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP1) inhibitory activity in the calcein AM accumulation assay and were found to be selective towards inhibition of BCRP. Substituents at position 2' and 4' on chalcone ring A were found to be essential for activity; additionally there was a great influence of substituents on ring B. Presence of 3,4-dimethoxy substitution on ring B was found to be optimal, while presence of 2- and 4-chloro substitution also showed a positive effect on BCRP inhibition.