Potent Inhibitor Of Dihydrofolate Reductase Research Articles

Quassinoids from Eurycoma longifolia as Potential Dihydrofolate Reductase Inhibitors: A Computational Study.

Quassinoids are degraded triterpene compounds that can be obtained from various species of the Simaroubaceae plant family, including Eurycoma longifolia. Quassinoids are the major compounds in E. longifolia, and they are known to have various medicinal potentials, such as anticancer and antimalarial properties. Dihydrofolate reductase (DHFR) was reported to be one of the important targets for certain anticancer and antimalarial drugs. Twelve quassinoids from E. longifolia were identified to have anticancer effects based on their IC50 values. This study aimed to evaluate the interactions of these twelve quassinoids with DHFR via Autodock 4.2 software and Biovia Discovery Studio Visualiser. Twelve quassinoids from E. longifolia and their interactions with DHFR were evaluated via Autodock 4.2 software and Biovia Discovery Studio Visualiser. Their drug-likeness and pharmacokinetic properties were also assessed using the ADMETlab 2.0 program. The molecular docking results showed that eleven quassinoids showed better docking scores than methotrexate, in which the binding energy (BE) of these quassinoids ranged from - 7.87 to -9.58 kcal/mol. Their inhibition constant (Ki) ranged from 0.095 to 1.71 μM. At the same time, the BE and Ki values for methotrexate were -7.80 kcal/mol and 1.64 μM, respectively. From the analysis, 6-dehydrolongilactone and eurycomalide B are among the twelve compounds that showed great potential as hit-to-lead compounds based on the docking score on DHFR, drug-likeness, and ADMET properties. These results suggest a great potential to pursue validation studies via in vitro and in vivo models.

Molecular Docking, In silico ADMET Study and Synthesis of Quinoline Derivatives as Dihydrofolate Reductase (DHFR) Inhibitors: A Solvent-free One-pot Green Approach Through Sonochemistry

Background: Quinoline derivatives have evinced their biological importance in targeting bacteria by inhibiting Dihydrofolate reductase. H2SO4 was successfully applied as an acid catalyst for a green, efficient, and one-pot solvent-free synthesis of quinoline derivatives using sonochemistry approach from various aromatic amines and glycerol with affording yield up to 96% within 6-10 min. Objective: In this study, the synthesis, characterization, and biological assessment of fifteen quinoline derivatives (1-15) as potential DHFR inhibitors were carried out. The target compounds were docked to study the molecular interactions and binding affinities with the 1DLS enzyme. Methods: The synthesized molecules were characterized using IR, MASS, and 1H and 13C NMR. The Insilico molecular docking study was carried out through target Human Dihydrofolate Reductase (DHFR) retrieved from a protein data bank having PDB ID: 1DLS and the antimicrobial activity of all synthesized compounds were tested against Human Dihydrofolate Reductase(DHFR) enzyme by using in-vitro DHFR assay kit. Results: The molecular docking results revealed that compounds 2 and 6 have the lowest binding energy and good binding affinity with the DHFR enzyme. In-silico ADMET predictions revealed that all bestscored compounds had good absorption and drug-like properties for potential use as DHFR inhibitors to treat bacterial infection. The in vitro studies revealed that compounds 2 and 6 show potent DFHR inhibitory activity against gram-positive and gram-negative with IC50 = 12.05 ± 1.55 μM and 10.04 ± 0.73 μM, respectively. While compounds 12, 13, and 15 exhibited moderate antimicrobial activity through DHFR inhibition with IC50= 16.33 ± 0.73 μM, 17.02 ± 1.55 μM, and 18.04 ± 1.05 μM, respectively. Conclusion: This environmentally benign sonochemistry-based approach for synthesizing quinoline derivatives could be affordable for large-scale production and become a potential lead candidate for developing a new quinoline-based antimicrobial agent.

Modeling of new potential inhibitors of dihydrofolate reductase based on 1,3,4-thiadiazole amidoalkyl derivatives

Derivatives of 1,3,4-thiadiazole are very important for medical chemistry and pharmacy as potential drug substances. In this work, we carried out molecular docking studies of amidoalkyl derivatives of 1,3,4-thiadiazole: N-(2,2,2-trichloro-1-((5-aryl-1,3,4-thiadiazol-2-yl)amino)ethyl)carboxamides and N-(2,2,2-trichloro-1-((5-(arylamino)-1,3,4-thiadiazol-2-yl)amino)ethyl)carboxamides with dihydrofolate reductase (DHFR). The AutoDock Vina program based on the PyRx 0.8 platform was used for docking. Before docking, the enzyme structure (PDB ID: 1DLS) was prepared using the Chimera 1.14 program, and the structures of potential inhibitors and reference preparations were optimized by the PM3 method in the ArgusLab 4.0.1 program. According to the results of molecular docking, the analyzed compounds effectively interact with the active site of DHFR. It is shown that the introduction of an NH group between the 1,3,4-thiadiazole and aromatic rings leads to stronger binding of ligands to DHFR. Based on the results of molecular docking, the following hit compounds were selected: 4-methyl-N-(2,2,2-trichloro-1-((5-(phenylamino)-1,3,4-thiadiazol-2-yl)amino)ethyl)benzamide and 4-methyl-N-(2,2,2-trichloro-1-((5-(p-tolylamino)-1,3,4-thiadiazol-2-yl)amino)ethyl)benzamide, which are superior to the reference compounds according to the strength of the formed complex.

Fighting Antibiotic Resistance: New Pyrimidine-Clubbed Benzimidazole Derivatives as Potential DHFR Inhibitors.

The present work describes the design and development of seventeen pyrimidine-clubbed benzimidazole derivatives as potential dihydrofolate reductase (DHFR) inhibitors. These compounds were filtered by using ADMET, drug-likeness characteristics calculations, and molecular docking experiments. Compounds 27, 29, 30, 33, 37, 38, and 41 were chosen for the synthesis based on the results of the in silico screening. Each of the synthesized compounds was tested for its in vitro antibacterial and antifungal activities using a variety of strains. All the compounds showed antibacterial properties against Gram-positive bacteria (Staphylococcus aureus and Staphylococcus pyogenes) as well as Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). Most of the compounds either had a higher potency than chloramphenicol or an equivalent potency to ciprofloxacin. Compounds 29 and 33 were effective against all the bacterial and fungal strains. Finally, the 1,2,3,4-tetrahydropyrimidine-2-thiol derivatives with a 6-chloro-2-(chloromethyl)-1H-benzo[d]imidazole moiety are potent enough to be considered a promising lead for the discovery of an effective antibacterial agent.

Design, synthesis and antitumor evaluation of novel pyrazolo[3,4-d]pyrimidines incorporating different amino acid conjugates as potential DHFR inhibitors

The present study aimed to investigate the antitumor effect of simultaneous inhibition of dihydrofolate reductase (DHFR) enzyme. We designed some novel pyrazolo[3,4-d]pyrimidines bearing different amino acid conjugates as efficient antifolate agents attributable to their structural similarity with methotrexate (MTX) and MTX-related antifolates. All compounds were tested to screen their enzymatic inhibition against DHFR compared with the reference drug MTX and for their in vitro antitumor cytotoxicity against six MTX-resistant cancer cell lines. The flow cytometry indicated that the most potent compound 7f arrested MCF-7 cells in the S-phase and induced apoptosis. Western blot for visualisation proved the ability of compound 7f to induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to diminish the expression of antiapoptotic Bcl-2 protein. Molecular modelling studies concluded that compound 7f displayed better binding energy than that of the normal ligand MTX.

Design, synthesis, and biological evaluation of some methyl 2-(1H-pyrazol-4-ylthio)-1,2,3,4-tetrahydro-6-methylpyrimidine-5-carboxylate derivatives as potential DHFR inhibitors

Drug-resistant bacteria pose an increasingly serious threat to mankind all over the world. However, the currently available clinical treatments do not meet the urgent demand. Therefore, it is desirable to find new targets and inhibitors to overcome the problems of antibiotic resistance. Dihydrofolate reductase (DHFR) is an important enzyme required to maintain bacterial growth, and hence inhibitors of DHFR have been proven as effective agents for treating bacterial infections. In the present work, we have designed some methyl 2-(1H-pyrazol-4-ylthio)-1,2,3,4-tetrahydro-6-methylpyrimidine-5-carboxylate derivatives as potential DHFR inhibitors through rational drug design approach. The designed derivatives were screened through Lipinski rule, Veber’s rule, ADMET analysis, drug-likeness properties, and molecular docking. All the compounds demonstrated more potent activity than Ampicillin against both gram-positive and gram-negative bacteria. Most of the compounds were more or equipotent than Chloramphenicol and Ciprofloxacin. Compound A7 was sensitive at 25 µg/mL against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus whereas compound A20 was sensitive to all gram +ve and –ve bacteria at same concentration. Compound A16 was sensitive at 50 µg/mL against all the bacteria. In antifungal activity, compound A7 exhibited MFCs of 100 µg/mL against Candida albicans, Aspergillus niger, and Aspergillus clavatus which is same as Nystatin.

Phytochemical constituents of Inula britannica as potential inhibitors of dihydrofolate reductase: A strategic approach against shigellosis

Shigella dysenteriae type 1 is considered as an epidemic in different developing countries, which is responsible for the most severe form of bacterial dysentery. It habitually can develop to the most severe form of dysentery with deadly complications. Development of drugs against this disease is still ongoing. Therefore, we used in silico studies to screen the Inula britannica phytocompounds that are used in traditional Chinese and Kampo Medicines and have activities against different diseases. Spinacetin, eupatin, chrysoeriol and diosmetin were successfully passed through the docking-based screening and absorption, distribution, metabolism, excretion and toxicity (ADMET) filtration. The estimated docking affinities of eupatin, diosmetin, chrysoeriol and spinacetin with Dihydrofolate reductase type 1 (DHFR-1), were −6.5, −6.5, −6.3 and −6.1 kcal/mol, respectively. Which were selected for further investigations based on their favorable ADME/Tox characteristics. Then, the 100 ns molecular dynamics (MD) simulations of apo DHFR, spinacetin-DHFR, eupatin-DHFR, chrysoeriol-DHFR and diosmetin-DHFR complexes were carried out. The RMSD fluctuations of the spinacetin, eupatin, chrysoeriol and diosmetin inside the binding site were explored. Subsequently, the effect of binding Spinacetin, eupatin, chrysoeriol and diosmetin upon the dynamic stability of protein was assessed. Additionally, Principal Component Analysis (PCA) and Hydrogen bond analysis was performed for the apo protein and the protein ligand complexes. The results revealed that chrysoeriol and eupatin has good inhibitory effects against DHFR-1 as treatment for Shigella dysenteriae type when compared to other compounds under study. Hence this study implies that eupatin and chrysoeriol are a significantly potential drug like molecule for the treatment of Shigellosis and must undergo validation through in vivo and in vitro experiments. Communicated by Ramaswamy H. Sarma

Structural Insights of Three 2,4-Disubstituted Dihydropyrimidine-5-carbonitriles as Potential Dihydrofolate Reductase Inhibitors

In this report, we describe the structural characterization of three 2,4-disubstituted-dihydropyrimidine-5-carbonitrile derivatives, namely 2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-4-propyl-1,6-dihydropyrimidine-5-carbonitrile 1, 4-(2-methylpropyl)-2-{[(4-nitrophenyl)methyl]sulfanyl}-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 2, and 2-[(2-ethoxyethyl)sulfanyl]-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile monohydrate 3. An X-ray diffraction analysis revealed that these compounds were crystallized in the centrosymmetric space groups and adopt an L-shaped conformation. One of the compounds (3) crystallized with a water molecule. A cyclic motif (R22(8)) mediated by N–H···O hydrogen bond was formed in compounds 1 and 2, whereas the corresponding motif was not favorable, due to the water molecule, in compound 3. The crystal packing of these compounds was analyzed based on energy frameworks performed at the B3LYP/6-31G(d,p) level of theory. Various inter-contacts were characterized using the Hirshfeld surface and its associated 2D-fingerprint plots. Furthermore, a molecular docking simulation was carried out to assess the inhibitory potential of the title compounds against the human dihydrofolate reductase (DHFR) enzyme.

Prospect into therapeutic potentials of Moringa oleifera phytocompounds against cancer upsurge: de novo synthesis of test compounds, molecular docking, and ADMET studies

BackgroundCancer chemotherapy is difficult because current medications for the treatment of cancer have been linked to a slew of side effects; as a result, researchers are tasked with developing greener cancer chemotherapies. Moringa oleifera has been reported with several bioactive compounds which confirm its application for various ailments by traditional practitioners. In this study, we aim to prospect the therapeutic potentials of M. oleifera phytocompounds against cancer proliferation as a step towards drug discovery using a computational approach. Target proteins: dihydrofolate reductase (DHFR) and B-Cell Lymphoid-2 (BCL-2), were retrieved from the RCSB PDB web server. Sixteen and five phytocompounds previously reported in M. oleifera leaves (ML) and seeds (MS), respectively, by gas chromatography–mass spectrometry were synthesized and used in the molecular docking study. For accurate prediction of binding sites of the target proteins; standard inhibitors, Methotrexate (MTX) for DHFR, and Venetoclax (VTC) for BCL-2, were docked together with the test compounds. We further predicted the ADMET profile of the potential inhibitors for an insight into their chance of success as candidates in drug discovery.ResultsResults for the binding affinities, docking poses, and the interactions showed that ML2, ML4-6, ML8-15, and MS1-5 are potential inhibitors of DHFR and BCL-2, respectively. In the ADMET profile, ML2 and ML4 showed the best drug-likeness by non-violation of Lipski Rule of Five. ML4-6, ML8, ML11, ML14-15, and MS1, MS3-5 exhibit high GI absorption; ML2, ML4-6, ML8, MS1, and MS5 are blood–brain barrier permeants. ML2, ML4, ML9, ML13, and MS2 do not interfere with any of the CYP450 isoforms. The toxicity profile showed that all the potential inhibitors are non-carcinogenic and non-hERG I (human ether-a-go-go related gene I) inhibitors. ML4, ML11, and MS4 are hepatotoxic and ML7, ML10, and MS4 are hERG II inhibitors. A plethora of insights on the toxic endpoints and lethal concentration values showed that ML5, ML13, and MS2 are comparatively less lethal than other potential inhibitors.ConclusionThis study has demonstrated that M. oleifera phytocompounds are potential inhibitors of the disease proteins involved in cancer proliferation, thus, an invaluable step toward the discovery of cancer chemotherapy with lesser limitations.

Integron mediated antimicrobial resistance in diarrheagenic Escherichia coli in children: in vitro and in silico analysis

The exchange of genes between bacterial chromosome and plasmid(s) and their integration into integrons are mainly responsible for acquisition and dissemination of antibiotic resistance. We investigated the role of integrons and their underlying molecular mechanisms leading to development of adaptability in E. coli and eventual resistance to antimicrobials. Escherichia coli isolates (n = 120); including 40 diarrheagenic isolates, an even number of isolates from cases other than diarrhea, and equal number of isolates from healthy children recovered from fresh stool samples were used for identification of integron genes and gene cassettes. The association of integrons with antibiotic resistance was assayed before phylogenetic analysis. DNA sequence analysis revealed class 1 and 2 integrons in 55.83% and 21.66% isolates, respectively. The integron presence was found significantly associated with the probability of antibiotic resistance in E. coli; the association being highest with class 1 integron. Modelling and molecular docking along with molecular dynamics simulation analyses found ceftriaxone and amoxicillin as potential inhibitors of dihydrofolate reductase (DHFR). The class 1 integrons of these pathogenic isolates can serve as prospective therapeutic targets using specific silencing strategies and combinational antimicrobial therapy. The findings may be useful for the development of a potent and versatile drug for DHFR inhibition.

Acetylshikonin isolated from Lithospermum erythrorhizon roots inhibits dihydrofolate reductase and hampers autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice

Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts’ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.

Quantitative analysis of hydrogen and chalcogen bonds in two pyrimidine-5-carbonitrile derivatives, potential DHFR inhibitors: an integrated crystallographic and theoretical study.

Two potential bioactive pyrimidine-5-carbonitrile derivatives have been synthesized and characterized by spectroscopic techniques (1H and 13C-NMR) and the three dimensional structures were elucidated by single crystal X-ray diffraction at low temperature (160 K). In both structures, the molecular conformation is locked by an intramolecular C–H⋯C interaction involving the cyano and CH of the thiophene and phenyl rings. The intermolecular interactions were analyzed in a qualitative manner based on the Hirshfeld surface and 2D-fingerprint plots. The results suggest that the phenyl and thiophene moieties have an effect on the crystal packing. For instance, the chalcogen bonds are only preferred in the thiophene derivative. However, both structures uses a common N–H⋯O hydrogen bond motif. Moreover, the structures of 1 and 2 display 1D isostructurality and molecular chains stabilize by intermolecular N–H⋯O and N–H⋯N hydrogen bonds. The nature and extent of different non-covalent interactions were further characterized by the topological parameters derived from the quantum theory of atoms-in-molecules approach. This analysis indicates that apart from N–H⋯O hydrogen bonds, other non-covalent interactions are closed-shell in nature. A strong and linear N–H⋯O hydrogen bond shows intermediate bonding character between shared and closed-shell interactions. The molecular docking analysis suggests that both compounds display potential inhibitory effect against the dihydrofolate reductase (DHFR) enzyme from humans and Staphylococcus aureus.

Comparative in-silico parmacokinetics and molecular docking study on gedunin isolated from Azadirachta indica, its modified derivatives and selected antifolate drugs as potential dihydrofolate reductase inhibitors of Plasmodium falciparum

Comparative in-silico parmacokinetics and molecular docking study on gedunin isolated from Azadirachta indica, its modified derivatives and selected antifolate drugs as potential dihydrofolate reductase inhibitors of Plasmodium falciparum

Comparative in-silico parmacokinetics and molecular docking study on gedunin isolated from Azadirachta indica, its modified derivatives and selected antifolate drugs as potential dihydrofolate reductase inhibitors of Plasmodium falciparum

Introduction: Malaria is one of the most common diseases that threaten many of the subtropical and tropical regions. Countries where the risk of transmission of malaria is at the high rate are over a hundred currently and these counties are being visited by over 125, 000, 000 international travelers on yearly basis. Materials and Methods: Chemical structures of ligands were drawn with the MarvinSketch software and converted into SMILES strings for the calculation of pharmacokinetic parameters. Results: The predicted binding energies between the three selected antifolate drugs, gedunin, its derivatives (C=O, C2H5, C3H6O2, C4H8O2, CONH2, NH2, OCH3, OH) and the Plasmodium falciparium DHFR enzyme were -8.0, -7.5, -8.0, -9.5, -9.0, -8.4, -8.9, -8.2, -8.9, -8.7, -8.3, -8.4 Kcal/mol respectively. Conclusion: The results from the experiment showed that gedunin and its modified derivatives might be better antimalarial agents than the antifolate drugs as revealed by the predicted binding energies between the target enzyme and the ligands.

Design, synthesis and biological evaluation of antimalarial activity of new derivatives of 2,4,6-s-triazine

Dihydrofolate reductase (DHFR) is an important enzyme for de novo synthesis of nucleotides in Plasmodium falciparum and it is essential for cell proliferation. DHFR is a well known antimalarial target for drugs like cycloguanil and pyrimethamine which target its inhibition for their pharmacological actions. However, the clinical efficacies of these antimalarial drugs have been compromising due to multiple mutations occurring in DHFR that lead to drug resistance. In this background, we have designed 22 s-triazine compounds using the best five parameters based 3D-QSAR model built by using genetic function approximation. In-silico designed compounds were further filtered to 6 compounds based upon their ADME properties, docking studies and predicted minimum inhibitory concentrations (MIC). Out of 6 compounds, 3 compounds were synthesized in good yield over 95% and characterized using IR, 1HNMR, 13CNMR and mass spectroscopic techniques. Parasitemia inhibition assay was used to evaluate the antimalarial activity of s-triazine compounds against 3D7 strain of P. falciparum. All the three compounds (7, 13 and 18) showed 30 times higher potency than cycloguanil (standard drug). It was observed that compound 18 was the most active while the compound 13 was the least active. On the closer inspection of physicochemical properties and SAR, it was observed that the presence of electron donating groups, number of hydrogen bond formation, lipophilicity of ligands and coulson charge of nitrogen atom present in the triazine ring enhances the DHFR inhibition significantly. This study will contribute to further endeavours of more potent DHFR inhibitors.

Schedule-Dependent Synergy Between Belinostat and Pralatrexate in T-and B-Cell Lymphoma Cells in Vitro

Pralatrexate (Folotyn; FOL) and belinostat (Beleodaq; BEL) are two new agents that have recently been registered for the treatment of patients with peripheral T-cell lymphoma (PTCL) and have also shown promising activity in other types of lymphoma. FOL is a folate analogue and a potent inhibitor of dihydrofolate reductase (DHFR), designed to accumulate in cancer cells preferentially via the reduced folate carrier (RFC) and retained therein via efficient polyglutamylation. Inhibition of DHFR leads to an imbalance of deoxynucleotides (e.g. depletion of dTTP and an increase in dUTP) resulting in DNA strand breaks and inhibition of DNA synthesis. BEL is a hydroxamic acid-based pan-histone deacetylase (HDAC) inhibitor that broadly inhibits all of the zinc-dependent HDAC enzymes, with high affinity for the Class I, II and IV isozymes. HDAC inhibition results in an alteration in the degree of histone and non-histone protein acetylation, which in turn affects transcription of genes essential in cellular proliferation, cell cycle and DNA repair. We investigated whether folate transporters other than RFC, i.e. folate receptor α (FRα) and the proton-coupled folate transporter (PCFT) could contribute to the efficacy of FOL. Moreover, we explored whether the toxicity of FOL can be controlled by levo-leucovorin (Fusilev), the natural stereoisomer of leucovorin, and whether in combination experiments BEL had the ability to potentiate the cytotoxicity of FOL.A panel of lymphoma cell lines was used for the combination studies including: the B-cell lymphoma cell lines SU-DHL-4, SU-DHL-5, HT, Jeko-1 and T-cell lymphoma cell lines Karpas-299 and Hut-78. RFC-mediated uptake efficiency of FOL was determined in competition uptake experiments with [3H] Methotrexate (MTX), revealing a 6-fold better RFC substrate affinity for FOL, and 2-fold better than levo-leucovorin. FOL displayed very poor substrate binding affinity for FRα (>100-fold lower than folic acid and > 10 lower than levo-leucovorin). FOL had a low substrate affinity for PCFT (>10-fold lower than folic acid and levo-leucovorin in [3H] leucovorin uptake competition experiments). Levo-leucovorin could completely protect toxicity by FOL, but had no effect on BEL toxicity. Sensitivity of lymphoma cell lines (IC50 concentrations after 72 hrs drug exposure) to FOL drug varied from 2.8 nM (Hut-78), 5.5 nM (SU-DHL4 and 5), 7.4 nM (HT) to 20 nM (Karpas-299 and Jeko-1) while IC50 values for BEL were in the range of 100 nM (SU-DLH-4 and 5, Jeko-1 and Hut-78) to 200 nM (Karpas-299 and HT).The interaction between BEL and FOL was studied using the median-drug effect analysis with Calcusyn software. At a fixed ratio between the drugs based on the IC50 concentration the average combination index (CI) for all the lymphoma cell lines revealed an additive effect (CI: all around 1.0). In two selected cell lines (SU-DHL-4 and HT) sequential exposure to the drugs (24 hr pretreatment with either BEL or FOL) followed by 48 hr to the combination, did not improve the results with CI values varying between 0.9 and 1.4. As an alternative approach a non-fixed ratio was used by exposing SU-DHL-4 and HT cells to IC25 concentrations of either BEL or FOL in combination with the other drug. Exposure to IC25 concentrations of FOL did not decrease the IC50 for BEL (CI around 1.2), but exposure to IC25 concentrations of BEL markedly increased the sensitivity to FOL as reflected by the low CIs varying from 0.40 to 0.66. Mechanistic studies focused on induction of apoptosis, showed cleavage of caspase 8 and 9 in HT and SU-DHL-4 cells for both drugs at their IC50s, being similar in the combination setting. Moreover, at these concentrations, the drugs were shown to confer an S-phase arrest.In conclusion, the combination of FOL and BEL showed additive activity in various lymphoma cell lines, while a schedule-dependent synergism was observed. Based on these data, proficient inhibition of HDAC activity by BEL holds promise in sensitization of tumor cells to FOL. Furthermore, toxicity of FOL could be completely protected by levo-leucovorin. DisclosuresReddy:spectrum: Employment, Equity Ownership.

Cheaper faster drug development validated by the repositioning of drugs against neglected tropical diseases.

There is an urgent need to make drug discovery cheaper and faster. This will enable the development of treatments for diseases currently neglected for economic reasons, such as tropical and orphan diseases, and generally increase the supply of new drugs. Here, we report the Robot Scientist ‘Eve’ designed to make drug discovery more economical. A Robot Scientist is a laboratory automation system that uses artificial intelligence (AI) techniques to discover scientific knowledge through cycles of experimentation. Eve integrates and automates library-screening, hit-confirmation, and lead generation through cycles of quantitative structure activity relationship learning and testing. Using econometric modelling we demonstrate that the use of AI to select compounds economically outperforms standard drug screening. For further efficiency Eve uses a standardized form of assay to compute Boolean functions of compound properties. These assays can be quickly and cheaply engineered using synthetic biology, enabling more targets to be assayed for a given budget. Eve has repositioned several drugs against specific targets in parasites that cause tropical diseases. One validated discovery is that the anti-cancer compound TNP-470 is a potent inhibitor of dihydrofolate reductase from the malaria-causing parasite Plasmodium vivax.

Sanguinarine suppresses basal-like breast cancer growth through dihydrofolate reductase inhibition

Basal-like breast cancer (BLBC) remains a great challenge because of its clinically aggressive nature and lack of effective targeted therapy. We analyzed the potential anti-neoplastic effects of sanguinarine, a natural benzophenanthridine alkaloid, against BLBC cells. Sanguinarine treatment resulted in a reduction of cell migration, in a dose-dependent inhibition of cell viability and in the induction of cell death by apoptosis in both human (MDA-MB-231 cells) and mouse (A17 cells) in vitro models of BLBC. In vivo experiments demonstrated that oral administration of sanguinarine reduced the development and growth of A17 transplantable tumors in FVB syngeneic mice. Western blotting analysis revealed that suppression of BLBC growth by sanguinarine was correlated with a concurrent upregulation of p27 and downregulation of cyclin D1 and with the inhibition of STAT3 activation. In addition, we identified sanguinarine as a potent inhibitor of dihydrofolate reductase (DHFR), able to impair enzyme activity even in methotrexate resistant MDA-MB-231 cells. These results provide evidence that sanguinarine is a promising anticancer drug for the treatment of BLBC.

In silico screening of chalcone derivatives as potential inhibitors of dihydrofolate reductase: Assessment using molecular docking, paired potential and molecular hydrophobic potential studies

ObjectivesEnzyme dihydro foliate reductase (DHFR) is involved in synthesis of DNA and consequently, has long been recognized to have utmost therapeutic significance, since its inactivation can be targeted in numerous infectious as well as noninfectious diseases. In the present studies molecular docking of chalcone derivatives with human as well as Mycobacterial DHFR, followed by paired potential and hydrophobic potential analysis were carried out to understand the novel chalcone–DHFR interactions. MethodsMolecular docking was carried out using GOLD and AutoDock software, paired potential analysis was performed employing on-line program DSX-ONLINE and molecular hydrophobic potential (MHP) analysis was done using web-based program PLATINUM. ResultsResults obtained from docking study, drug score potential and MHP analysis coincide with experimental findings. Molecular property analysis indicates that given compounds follows Lipinski's rule of five. Compound number 1 exhibited best binding energy (−8.02 kcal/mol) in human DHFR while compound number 6 (−7.36 kcal/mol), 9 (−7.32 kcal/mol), 10 (−7.31 kcal/mol) and 11 (−8.25 kcal/mol) demonstrated favorable binding score in Mycobacterial DHFR. ConclusionsPer atom score contribution of chalcone derivatives obtained by paired potential analysis indicate participation of conserved as well as few new residues are expected to be involved in inhibition of DHFR. MHP analysis of chalcone–DHFR complexes revealed important role of hydrophobic contact in inhibition; additionally, individual chemical scaffold on chalcone derivatives that contribute in lipophilicity has been identified. This data is expected to be further explored for the design and development of novel class of DHFR inhibitors using chalcone scaffold.

Kinetic and Structural Analysis for Potent Antifolate Inhibition of Pneumocystis jirovecii, Pneumocystis carinii, and Human Dihydrofolate Reductases and Their Active-Site Variants

A major concern of immunocompromised patients, in particular those with AIDS, is susceptibility to infection caused by opportunistic pathogens such as Pneumocystis jirovecii, which is a leading cause of pneumonia in immunocompromised patients. We report the first kinetic and structural data for 2,4-diamino-6-[(2',5'-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine (OAAG324), a potent inhibitor of dihydrofolate reductase (DHFR) from P. jirovecii (pjDHFR), and also for trimethoprim (TMP) and methotrexate (MTX) with pjDHFR, Pneumocystis carinii DHFR (pcDHFR), and human DHFR (hDHFR). OAAG324 shows a 9.0-fold selectivity for pjDHFR (Ki, 2.7 nM) compared to its selectivity for hDHFR (Ki, 24.4 nM), whereas there is only a 2.3-fold selectivity for pcDHFR (Ki, 6.3 nM). In order to understand the determinants of inhibitory potency, active-site mutations of pj-, pc-, and hDHFR were explored to make these enzymes more like each other. The most unexpected observations were that the variant pcDHFR forms with K37Q and K37Q/F69N mutations, which made the enzyme more like the human form, also made these enzymes more sensitive to the inhibitory activity of OAAG324, with Ki values of 0.26 and 0.71 nM, respectively. A similar gain in sensitivity was also observed for the hDHFR N64F variant, which showed a lower Ki value (0.58 nM) than native hDHFR, pcDHFR, or pjDHFR. Structural data are reported for complexes of OAAG324 with hDHFR and its Q35K and Q35S/N64F variants and for the complex of the K37S/F69N variant of pcDHFR with TMP. These results provide useful insight into the role of these residues in the optimization of highly selective inhibitors of DHFR against the opportunistic pathogen P. jirovecii.