Rosaria Luciani | Università degli studi di Modena e Reggio Emilia (original) (raw)
Papers by Rosaria Luciani
Composti destabilizzanti l\u2019omodimero timidilato sintas
Journal of Medicinal Chemistry
The optimization of compounds with multiple targets is a difficult multidimensional problem in th... more The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure-activity relationship for multitarget inhibition. The approach yielded compounds showing subnanomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.
Pharmaceuticals, 2021
Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screenin... more Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screening by GlaxoSmithKline (Tres Cantos Anti-Kinetoplastid Screening, TCAKS) were exploited for the discovery of a novel core structure inspiring new treatments of parasitic diseases targeting the trypansosmatidic pteridine reductase 1 (PTR1) and dihydrofolate reductase (DHFR) enzymes. In total, 592 compounds were tested through medium-throughput screening assays. A subset of 14 compounds successfully inhibited the enzyme activity in the low micromolar range of at least one of the enzymes from both Trypanosoma brucei and Lesihmania major parasites (pan-inhibitors), or from both PTR1 and DHFR-TS of the same parasite (dual inhibitors). Molecular docking studies of the protein–ligand interaction focused on new scaffolds not reproducing the well-known antifolate core clearly explaining the experimental data. TCMDC-143249, classified as a benzenesulfonamide derivative by the QikProp descriptor tool...
The optimization of compounds with multiple targets in the drug discovery cycle is a difficult mu... more The optimization of compounds with multiple targets in the drug discovery cycle is a difficult multidimensional problem. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Efficient microwave-assisted synthesis of pteridines along with iterations of crystallographic structure determination were used to validate computational docking predictions and support derivation of a structure-activity relationship for multitarget inhibition. This approach yielded compounds showing picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, along with selective submicromolar inhibition of parasitic dihydrofolate reductase (DHFR). Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for t...
Drugs that target human thymidylate synthase (hTS) are widely used in anti-cancer therapy. Howeve... more Drugs that target human thymidylate synthase (hTS) are widely used in anti-cancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces its over-expression and the development of drug resistance. We thus pursued an alternative strategy that led to the discovery of TS-dimer disrupters that bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. We performed a structural, spectroscopic and kinetic investigation of the effects of these small molecules andthe best one, E7, accelerates the proteasomal degradation of hTS in cancer cells. E7 showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-r...
Molecules, 2019
Thymidylate synthase (TS) is a prominent drug target for different cancer types. However, the pro... more Thymidylate synthase (TS) is a prominent drug target for different cancer types. However, the prolonged use of its classical inhibitors, substrate analogs that bind at the active site, leads to TS overexpression and drug resistance in the clinic. In the effort to identify anti-TS drugs with new modes of action and able to overcome platinum drug resistance in ovarian cancer, octapeptides with a new allosteric inhibition mechanism were identified as cancer cell growth inhibitors that do not cause TS overexpression. To improve the biological properties, 10 cyclic peptides (cPs) were designed from the lead peptides and synthesized. The cPs were screened for the ability to inhibit recombinant human thymidylate synthase (hTS), and peptide 7 was found to act as an allosteric inhibitor more potent than its parent open-chain peptide [Pro3]LR. In cytotoxicity studies on three human ovarian cancer cell lines, IGROV-1, A2780, and A2780/CP, peptide 5 and two other cPs, including 7, showed IC50 v...
Bioorganic & Medicinal Chemistry Letters, 2018
Highlights Analogues of known Hsp90 inhibitors with a permanent or ionizable cationic head. I... more Highlights Analogues of known Hsp90 inhibitors with a permanent or ionizable cationic head. Inhibition of TRAP1 ATPase activity and detection in mitochondrial cell fraction. Best apoptotic and growth inhibition activities of guanidine bearing derivative. Similar affinities of most active compound for Hsp90 and TRAP1 binding.
ACS Medicinal Chemistry Letters, 2019
In vitro evaluation of activity against T. brucei The efficacy of compounds against T. brucei blo... more In vitro evaluation of activity against T. brucei The efficacy of compounds against T. brucei bloodstream forms was evaluated using a modified resazurin-based assay previously described in literature. 34 The reported EC50 and NOAEL are the arithmetic average of at least two independent determinations obtained in triplicate. Cytotoxicity assessment against THP-1 macrophages The effect of compounds 1-21 on THP-1-derived macrophages was assessed by the colorimetric MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. hERG assay This assay made use of Invitrogen's Predictor™ hERG Fluorescence Polarisation Assay. The assay uses a membrane fraction containing hERG channel (Predictor™ hERG Membrane) and a highaffinity red fluorescent hERG channel ligand, or "tracer" (Predictor™ hERG Tracer Red), whose displacement by test compounds can be determined in a homogenous, Fluorescence Polarisation (FP) based format. 15 Cytochrome P450 1A2, 2C9, 2C19, 2D6 and 3A4 assays. These assays made use of the Promega P450-Glo™assayplatform. Each CYP450 assay made use of microsomal preparations of cytochromes from baculovirus infected insect cells. Action of the CYP450 enzymes upon each substrate ultimately resulted in the generation of light and a decrease in this was indicative of inhibition of the enzymes. 15 Cytotoxicity assay against A549 cells The assays were performed using the Cell Titer-Glo assay from Promega. The assay detects cellular ATP content with the amount of ATP being directly proportional to the number of the present cells. The A549 cells were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and WI38 cells were obtained from ATCC (ATCC ® CCL-75™) and were grown in DMEM with FCS (10 % v/v), streptomycin (100 μg/ml) and penicillin G (100 U/ml). 15 Assessment of mitochondrial toxicity This assay made use of MitoTracker ® Red chloromethyl-X-rosamine (CMXRos) uptake and High Content Imaging to monitor compound mediated mitochondrial toxicity in the 786-O (renal carcinoma) cell line. Cells were maintained using RPMI-1640 medium containing 2 mM glutamine, FCS (10 % v/v), streptomycin (100 μg/ml) and penicillin G (100 U/ml). 15 Pharmacokinetics of compound 1 For pharmacokinetic studies BALB/c mice were treated with compound 1 administered (8 % DMSO) 1 mg/kg IV or 20 mg/kg per os. Plasma samples were analyzed by LC-MS/MS. Chromatographic separation was carried out using a Shimadzu LC system consisting of two pumps, column oven, degasser and autosampler. Attached to this system was an analytical column (C18, Gemini 5 µm 110 A Phenomenex, 150 x 2 mm). The HPLC system was connected to a triple-quadrupole mass spectrometer equipped with a turboionspray source operated with unit resolution in the positive ion mode (ESI-QQQMS, Shimadzu LCMS-30). Under these conditions a retention time of approximately S3 1.2 minutes for the molecule was obtained. Mobile phase consisted of a mixture (80:20) of acetonitrile:purified water flow at a 0.5 ml/min rate in isocratic mode. Run time was 5 minutes and the injection volume was 50 μl. The mass transition of m/z was: Quantifier (m/z): 313.3 > 184 CE:-44; Qualifier (m/z): 313.3 >227.05 CE:-33. Plasma samples of mice were obtained by serial sampling from submandibular vein and stored at-20 ºC until analyzed. Spectroscopic investigation The absorption spectra were measured with a Cary 100 UV-visible spectrophotometer, the fluorescence emission spectra with a Spex Jobin-Yvon FluoroMax3 spectrofluorometer. 1x0.4 cm2 quartz cuvettes were used. HSA (Sigma, ≥ 97%) was used as such. Solutions of compound 1 in phosphate buffer at pH 8 were obtained from mother solutions in dimethylsulfoxide. Ethics Statement The experimental design and housing conditions at UCM were approved by the Committee of Animal Experimentation (Universidad Complutense de Madrid) and regional authorities (Community of Madrid) (Ref. PROEX 169/15). Experiments were carried out at the animal house with official identification code ES280790001164 following the 3Rs principles. Animal handling and sampling were performed by trained and officially qualified personnel. (34) Bowling, T.; Mercer, L.; Don, R.; Jacobs, R.; Nare, B. Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasis. Int
Molecules, 2019
Thymidylate synthase (TS) is an enzyme of paramount importance as it provides the only de novo so... more Thymidylate synthase (TS) is an enzyme of paramount importance as it provides the only de novo source of deoxy-thymidine monophosphate (dTMP). dTMP, essential for DNA synthesis, is produced by the TS-catalyzed reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) using N5,N10-methylenetetrahydrofolate (mTHF) as a cofactor. TS is ubiquitous and a validated drug target. TS enzymes from different organisms differ in sequence and structure, but are all obligate homodimers. The structural and mechanistic differences between the human and bacterial enzymes are exploitable to obtain selective inhibitors of bacterial TSs that can enrich the currently available therapeutic tools against bacterial infections. Enterococcus faecalis is a pathogen fully dependent on TS for dTMP synthesis. In this study, we present four new crystal structures of Enterococcus faecalis and human TSs in complex with either the substrate dUMP or the inhibitor FdUMP. The results provide new clues about the ...
Journal of medicinal chemistry, Jan 23, 2018
LR and [D-Gln4]LR peptides bind the monomer-monomer interface of human thymidylate synthase and i... more LR and [D-Gln4]LR peptides bind the monomer-monomer interface of human thymidylate synthase and inhibit cancer cell growth. Here, proline-mutated LR peptides were synthesized. Molecular dynamics calculations and circular dichroism spectra have provided a consistent picture of the conformational propensities of the [Pron]-peptides. [Pro3]LR and [Pro4]LR show improved cell growth inhibition and similar intracellular protein modulation than LR. These represent a step forward to the identification of more rigid and metabolically stable peptides.
European journal of medicinal chemistry, Jan 25, 2018
Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have co... more Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have considered the thiadiazoles opened forms and reaction intermediates, thiosemicarbazones, as compounds of interest for phenotypic screening against Trypanosoma brucei (Tb), intracellular amastigote form of Leishmania infantum (Li) and Trypanosoma cruzi (Tc). Similar compounds have already shown interesting activity against the same organisms. The compounds were particularly effective against T. brucei and T. cruzi. Among the 28 synthesized compounds, the best one was (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene) hydrazinecarbothioamide (A14) yielding a comparable anti-parasitic activity against the three parasitic species (TbEC = 2.31 μM, LiEC = 6.14 μM, TcEC = 1.31 μM) and a Selectivity Index higher than 10 with respect to human macrophages, therefore showing a pan-anti-trypanosomatidic activity. (E)-2-((3'.4'-dimethoxy-[1.1'-biphenyl]-3-yl)methyle ne) hydrazinecarbothioamide ...
SPIE Proceedings, 2017
In veterinary practice antibiotics are used and abused all over the world both to treat diseases ... more In veterinary practice antibiotics are used and abused all over the world both to treat diseases and prevent infections and, to increase feed efficiency thus promoting growth in food producing animals. With more than 30 years history, β−lactam antibiotics are nowadays one of the most important group of antibiotics in veterinary medicine. In this paper we investigate the possibility to realize a measurement method suitable for field testing and we propose a competitive biosensor based on a relatively low-cost chromogenic cephalosporin (CENTA) for the quantitative analysis of β−lactams concentration in milk. In particular, the reported preliminary study has been focused on cloxacillin and the reported results indicates that the method is potentially able to detect cloxacillin at a concentration of about one order of magnitude lower than the maximal residue limits (MRL) set by the European Commission.
ACS Omega, 2017
Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We ... more Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme-and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC 50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC 50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.
Acta Crystallographica Section A Foundations of Crystallography, 2010
Journal of medicinal chemistry, Oct 13, 2016
Thymidylate synthase X (ThyX) represents an attractive target for tuberculosis drug discovery. He... more Thymidylate synthase X (ThyX) represents an attractive target for tuberculosis drug discovery. Herein, we selected 16 compounds through a virtual screening approach. We solved the first X-ray crystal structure of Thermatoga maritima (Tm) ThyX in complex with a nonsubstrate analog inhibitor. Given the active site similarities between Mycobacterium tuberculosis ThyX (Mtb-ThyX) and Tm-ThyX, our crystal structure paves the way for a structure-based design of novel antimycobacterial compounds. The 1H-imidazo[4,5-d]pyridazine was identified as scaffold for the development of Mtb-ThyX inhibitors.
European Journal of Medicinal Chemistry, 2016
Infections caused by Enterococcus faecalis (Ef) represent nowadays a relevant health problem. We ... more Infections caused by Enterococcus faecalis (Ef) represent nowadays a relevant health problem. We selected Thymidylate synthase (TS) from this organism as a potential specific target for antibacterial therapy. We have previously demonstrated that species-specific inhibition of the protein can be achieved despite the relatively high structural similarity among bacterial TSs and human TS. We had previously obtained the EfTS crystal structure of the protein in complex with the metabolite 5-formyl-tetrahydrofolate (5-FTHF) suggesting the protein role as metabolite reservoir; however, protein-inhibitors complexes were still missing. In the present work we identified some inhibitors bearing the phthalimidic core from our in-house library and we performed crystallographic screening towards EfTS. We obtained two X-ray crystallographic structures: the first with a weak phthalimidic inhibitor bound in one subunit and 5hydroxymethylene-6-hydrofolic acid (5-HMHF) in the other subunit; a second X-ray structure complex with methotrexate. The structural information achieved confirm the role of EfTS as an enzyme involved in the folate pool system and provide a structural basis for structure-based drug design. 40 41 42 43 44 45 46 47 48
ChemMedChem, Aug 12, 2016
Repurposing and repositioning drugs has become a frequently pursued and successful strategy in th... more Repurposing and repositioning drugs has become a frequently pursued and successful strategy in the current era, as new chemical entities are increasingly difficult to find and get approved. Herein we report an integrated BioGPS/FLAPdock pipeline for rapid and effective off-target identification and drug repurposing. Our method is based on the structural and chemical properties of protein binding sites, that is, the ligand image, encoded in the GRID molecular interaction fields (MIFs). Protein similarity is disclosed through the BioGPS algorithm by measuring the pockets' overlap according to which pockets are clustered. Co-crystallized and known ligands can be cross-docked among similar targets, selected for subsequent in vitro binding experiments, and possibly improved for inhibitory potency. We used human thymidylate synthase (TS) as a test case and searched the entire RCSB Protein Data Bank (PDB) for similar target pockets. We chose casein kinase IIα as a control and tested a ...
Journal of Medicinal Chemistry, 2016
Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a librar... more Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC 50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure−activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.
Scientific reports, Jan 2, 2016
Demonstrating a candidate drug's interaction with its target protein in live cells is of pivo... more Demonstrating a candidate drug's interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new m...
Composti destabilizzanti l\u2019omodimero timidilato sintas
Journal of Medicinal Chemistry
The optimization of compounds with multiple targets is a difficult multidimensional problem in th... more The optimization of compounds with multiple targets is a difficult multidimensional problem in the drug discovery cycle. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Computational fragment-based design of novel pteridine derivatives along with iterations of crystallographic structure determination allowed for the derivation of a structure-activity relationship for multitarget inhibition. The approach yielded compounds showing subnanomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, and selective submicromolar inhibition of parasite dihydrofolate reductase (DHFR) versus human DHFR. Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for the further development of pteridine-based compounds, and we expect our multitarget approach to be generally applicable to the design and optimization of anti-infective agents.
Pharmaceuticals, 2021
Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screenin... more Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screening by GlaxoSmithKline (Tres Cantos Anti-Kinetoplastid Screening, TCAKS) were exploited for the discovery of a novel core structure inspiring new treatments of parasitic diseases targeting the trypansosmatidic pteridine reductase 1 (PTR1) and dihydrofolate reductase (DHFR) enzymes. In total, 592 compounds were tested through medium-throughput screening assays. A subset of 14 compounds successfully inhibited the enzyme activity in the low micromolar range of at least one of the enzymes from both Trypanosoma brucei and Lesihmania major parasites (pan-inhibitors), or from both PTR1 and DHFR-TS of the same parasite (dual inhibitors). Molecular docking studies of the protein–ligand interaction focused on new scaffolds not reproducing the well-known antifolate core clearly explaining the experimental data. TCMDC-143249, classified as a benzenesulfonamide derivative by the QikProp descriptor tool...
The optimization of compounds with multiple targets in the drug discovery cycle is a difficult mu... more The optimization of compounds with multiple targets in the drug discovery cycle is a difficult multidimensional problem. Here, we present a systematic, multidisciplinary approach to the development of selective anti-parasitic compounds. Efficient microwave-assisted synthesis of pteridines along with iterations of crystallographic structure determination were used to validate computational docking predictions and support derivation of a structure-activity relationship for multitarget inhibition. This approach yielded compounds showing picomolar inhibition of T. brucei pteridine reductase 1 (PTR1), nanomolar inhibition of L. major PTR1, along with selective submicromolar inhibition of parasitic dihydrofolate reductase (DHFR). Moreover, by combining design for polypharmacology with a property-based on-parasite optimization, we found three compounds that exhibited micromolar EC50 values against T. brucei brucei, whilst retaining their target inhibition. Our results provide a basis for t...
Drugs that target human thymidylate synthase (hTS) are widely used in anti-cancer therapy. Howeve... more Drugs that target human thymidylate synthase (hTS) are widely used in anti-cancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces its over-expression and the development of drug resistance. We thus pursued an alternative strategy that led to the discovery of TS-dimer disrupters that bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. We performed a structural, spectroscopic and kinetic investigation of the effects of these small molecules andthe best one, E7, accelerates the proteasomal degradation of hTS in cancer cells. E7 showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-r...
Molecules, 2019
Thymidylate synthase (TS) is a prominent drug target for different cancer types. However, the pro... more Thymidylate synthase (TS) is a prominent drug target for different cancer types. However, the prolonged use of its classical inhibitors, substrate analogs that bind at the active site, leads to TS overexpression and drug resistance in the clinic. In the effort to identify anti-TS drugs with new modes of action and able to overcome platinum drug resistance in ovarian cancer, octapeptides with a new allosteric inhibition mechanism were identified as cancer cell growth inhibitors that do not cause TS overexpression. To improve the biological properties, 10 cyclic peptides (cPs) were designed from the lead peptides and synthesized. The cPs were screened for the ability to inhibit recombinant human thymidylate synthase (hTS), and peptide 7 was found to act as an allosteric inhibitor more potent than its parent open-chain peptide [Pro3]LR. In cytotoxicity studies on three human ovarian cancer cell lines, IGROV-1, A2780, and A2780/CP, peptide 5 and two other cPs, including 7, showed IC50 v...
Bioorganic & Medicinal Chemistry Letters, 2018
Highlights Analogues of known Hsp90 inhibitors with a permanent or ionizable cationic head. I... more Highlights Analogues of known Hsp90 inhibitors with a permanent or ionizable cationic head. Inhibition of TRAP1 ATPase activity and detection in mitochondrial cell fraction. Best apoptotic and growth inhibition activities of guanidine bearing derivative. Similar affinities of most active compound for Hsp90 and TRAP1 binding.
ACS Medicinal Chemistry Letters, 2019
In vitro evaluation of activity against T. brucei The efficacy of compounds against T. brucei blo... more In vitro evaluation of activity against T. brucei The efficacy of compounds against T. brucei bloodstream forms was evaluated using a modified resazurin-based assay previously described in literature. 34 The reported EC50 and NOAEL are the arithmetic average of at least two independent determinations obtained in triplicate. Cytotoxicity assessment against THP-1 macrophages The effect of compounds 1-21 on THP-1-derived macrophages was assessed by the colorimetric MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. hERG assay This assay made use of Invitrogen's Predictor™ hERG Fluorescence Polarisation Assay. The assay uses a membrane fraction containing hERG channel (Predictor™ hERG Membrane) and a highaffinity red fluorescent hERG channel ligand, or "tracer" (Predictor™ hERG Tracer Red), whose displacement by test compounds can be determined in a homogenous, Fluorescence Polarisation (FP) based format. 15 Cytochrome P450 1A2, 2C9, 2C19, 2D6 and 3A4 assays. These assays made use of the Promega P450-Glo™assayplatform. Each CYP450 assay made use of microsomal preparations of cytochromes from baculovirus infected insect cells. Action of the CYP450 enzymes upon each substrate ultimately resulted in the generation of light and a decrease in this was indicative of inhibition of the enzymes. 15 Cytotoxicity assay against A549 cells The assays were performed using the Cell Titer-Glo assay from Promega. The assay detects cellular ATP content with the amount of ATP being directly proportional to the number of the present cells. The A549 cells were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and WI38 cells were obtained from ATCC (ATCC ® CCL-75™) and were grown in DMEM with FCS (10 % v/v), streptomycin (100 μg/ml) and penicillin G (100 U/ml). 15 Assessment of mitochondrial toxicity This assay made use of MitoTracker ® Red chloromethyl-X-rosamine (CMXRos) uptake and High Content Imaging to monitor compound mediated mitochondrial toxicity in the 786-O (renal carcinoma) cell line. Cells were maintained using RPMI-1640 medium containing 2 mM glutamine, FCS (10 % v/v), streptomycin (100 μg/ml) and penicillin G (100 U/ml). 15 Pharmacokinetics of compound 1 For pharmacokinetic studies BALB/c mice were treated with compound 1 administered (8 % DMSO) 1 mg/kg IV or 20 mg/kg per os. Plasma samples were analyzed by LC-MS/MS. Chromatographic separation was carried out using a Shimadzu LC system consisting of two pumps, column oven, degasser and autosampler. Attached to this system was an analytical column (C18, Gemini 5 µm 110 A Phenomenex, 150 x 2 mm). The HPLC system was connected to a triple-quadrupole mass spectrometer equipped with a turboionspray source operated with unit resolution in the positive ion mode (ESI-QQQMS, Shimadzu LCMS-30). Under these conditions a retention time of approximately S3 1.2 minutes for the molecule was obtained. Mobile phase consisted of a mixture (80:20) of acetonitrile:purified water flow at a 0.5 ml/min rate in isocratic mode. Run time was 5 minutes and the injection volume was 50 μl. The mass transition of m/z was: Quantifier (m/z): 313.3 > 184 CE:-44; Qualifier (m/z): 313.3 >227.05 CE:-33. Plasma samples of mice were obtained by serial sampling from submandibular vein and stored at-20 ºC until analyzed. Spectroscopic investigation The absorption spectra were measured with a Cary 100 UV-visible spectrophotometer, the fluorescence emission spectra with a Spex Jobin-Yvon FluoroMax3 spectrofluorometer. 1x0.4 cm2 quartz cuvettes were used. HSA (Sigma, ≥ 97%) was used as such. Solutions of compound 1 in phosphate buffer at pH 8 were obtained from mother solutions in dimethylsulfoxide. Ethics Statement The experimental design and housing conditions at UCM were approved by the Committee of Animal Experimentation (Universidad Complutense de Madrid) and regional authorities (Community of Madrid) (Ref. PROEX 169/15). Experiments were carried out at the animal house with official identification code ES280790001164 following the 3Rs principles. Animal handling and sampling were performed by trained and officially qualified personnel. (34) Bowling, T.; Mercer, L.; Don, R.; Jacobs, R.; Nare, B. Application of a resazurin-based high-throughput screening assay for the identification and progression of new treatments for human African trypanosomiasis. Int
Molecules, 2019
Thymidylate synthase (TS) is an enzyme of paramount importance as it provides the only de novo so... more Thymidylate synthase (TS) is an enzyme of paramount importance as it provides the only de novo source of deoxy-thymidine monophosphate (dTMP). dTMP, essential for DNA synthesis, is produced by the TS-catalyzed reductive methylation of 2′-deoxyuridine-5′-monophosphate (dUMP) using N5,N10-methylenetetrahydrofolate (mTHF) as a cofactor. TS is ubiquitous and a validated drug target. TS enzymes from different organisms differ in sequence and structure, but are all obligate homodimers. The structural and mechanistic differences between the human and bacterial enzymes are exploitable to obtain selective inhibitors of bacterial TSs that can enrich the currently available therapeutic tools against bacterial infections. Enterococcus faecalis is a pathogen fully dependent on TS for dTMP synthesis. In this study, we present four new crystal structures of Enterococcus faecalis and human TSs in complex with either the substrate dUMP or the inhibitor FdUMP. The results provide new clues about the ...
Journal of medicinal chemistry, Jan 23, 2018
LR and [D-Gln4]LR peptides bind the monomer-monomer interface of human thymidylate synthase and i... more LR and [D-Gln4]LR peptides bind the monomer-monomer interface of human thymidylate synthase and inhibit cancer cell growth. Here, proline-mutated LR peptides were synthesized. Molecular dynamics calculations and circular dichroism spectra have provided a consistent picture of the conformational propensities of the [Pron]-peptides. [Pro3]LR and [Pro4]LR show improved cell growth inhibition and similar intracellular protein modulation than LR. These represent a step forward to the identification of more rigid and metabolically stable peptides.
European journal of medicinal chemistry, Jan 25, 2018
Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have co... more Basing on a library of thiadiazole derivatives showing anti-trypanosomatidic activity, we have considered the thiadiazoles opened forms and reaction intermediates, thiosemicarbazones, as compounds of interest for phenotypic screening against Trypanosoma brucei (Tb), intracellular amastigote form of Leishmania infantum (Li) and Trypanosoma cruzi (Tc). Similar compounds have already shown interesting activity against the same organisms. The compounds were particularly effective against T. brucei and T. cruzi. Among the 28 synthesized compounds, the best one was (E)-2-(4-((3.4-dichlorobenzyl)oxy)benzylidene) hydrazinecarbothioamide (A14) yielding a comparable anti-parasitic activity against the three parasitic species (TbEC = 2.31 μM, LiEC = 6.14 μM, TcEC = 1.31 μM) and a Selectivity Index higher than 10 with respect to human macrophages, therefore showing a pan-anti-trypanosomatidic activity. (E)-2-((3'.4'-dimethoxy-[1.1'-biphenyl]-3-yl)methyle ne) hydrazinecarbothioamide ...
SPIE Proceedings, 2017
In veterinary practice antibiotics are used and abused all over the world both to treat diseases ... more In veterinary practice antibiotics are used and abused all over the world both to treat diseases and prevent infections and, to increase feed efficiency thus promoting growth in food producing animals. With more than 30 years history, β−lactam antibiotics are nowadays one of the most important group of antibiotics in veterinary medicine. In this paper we investigate the possibility to realize a measurement method suitable for field testing and we propose a competitive biosensor based on a relatively low-cost chromogenic cephalosporin (CENTA) for the quantitative analysis of β−lactams concentration in milk. In particular, the reported preliminary study has been focused on cloxacillin and the reported results indicates that the method is potentially able to detect cloxacillin at a concentration of about one order of magnitude lower than the maximal residue limits (MRL) set by the European Commission.
ACS Omega, 2017
Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We ... more Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme-and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC 50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC 50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.
Acta Crystallographica Section A Foundations of Crystallography, 2010
Journal of medicinal chemistry, Oct 13, 2016
Thymidylate synthase X (ThyX) represents an attractive target for tuberculosis drug discovery. He... more Thymidylate synthase X (ThyX) represents an attractive target for tuberculosis drug discovery. Herein, we selected 16 compounds through a virtual screening approach. We solved the first X-ray crystal structure of Thermatoga maritima (Tm) ThyX in complex with a nonsubstrate analog inhibitor. Given the active site similarities between Mycobacterium tuberculosis ThyX (Mtb-ThyX) and Tm-ThyX, our crystal structure paves the way for a structure-based design of novel antimycobacterial compounds. The 1H-imidazo[4,5-d]pyridazine was identified as scaffold for the development of Mtb-ThyX inhibitors.
European Journal of Medicinal Chemistry, 2016
Infections caused by Enterococcus faecalis (Ef) represent nowadays a relevant health problem. We ... more Infections caused by Enterococcus faecalis (Ef) represent nowadays a relevant health problem. We selected Thymidylate synthase (TS) from this organism as a potential specific target for antibacterial therapy. We have previously demonstrated that species-specific inhibition of the protein can be achieved despite the relatively high structural similarity among bacterial TSs and human TS. We had previously obtained the EfTS crystal structure of the protein in complex with the metabolite 5-formyl-tetrahydrofolate (5-FTHF) suggesting the protein role as metabolite reservoir; however, protein-inhibitors complexes were still missing. In the present work we identified some inhibitors bearing the phthalimidic core from our in-house library and we performed crystallographic screening towards EfTS. We obtained two X-ray crystallographic structures: the first with a weak phthalimidic inhibitor bound in one subunit and 5hydroxymethylene-6-hydrofolic acid (5-HMHF) in the other subunit; a second X-ray structure complex with methotrexate. The structural information achieved confirm the role of EfTS as an enzyme involved in the folate pool system and provide a structural basis for structure-based drug design. 40 41 42 43 44 45 46 47 48
ChemMedChem, Aug 12, 2016
Repurposing and repositioning drugs has become a frequently pursued and successful strategy in th... more Repurposing and repositioning drugs has become a frequently pursued and successful strategy in the current era, as new chemical entities are increasingly difficult to find and get approved. Herein we report an integrated BioGPS/FLAPdock pipeline for rapid and effective off-target identification and drug repurposing. Our method is based on the structural and chemical properties of protein binding sites, that is, the ligand image, encoded in the GRID molecular interaction fields (MIFs). Protein similarity is disclosed through the BioGPS algorithm by measuring the pockets' overlap according to which pockets are clustered. Co-crystallized and known ligands can be cross-docked among similar targets, selected for subsequent in vitro binding experiments, and possibly improved for inhibitory potency. We used human thymidylate synthase (TS) as a test case and searched the entire RCSB Protein Data Bank (PDB) for similar target pockets. We chose casein kinase IIα as a control and tested a ...
Journal of Medicinal Chemistry, 2016
Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a librar... more Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC 50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure−activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.
Scientific reports, Jan 2, 2016
Demonstrating a candidate drug's interaction with its target protein in live cells is of pivo... more Demonstrating a candidate drug's interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new m...