Kinetic and structural studies of Mycobacterium tuberculosis dihydroorotate dehydrogenase reveal new insights into class 2 DHODH inhibition.

Tuberculosis (TB) is a leading cause of death worldwide. TB represents a serious public health threat, and it is characterized by high transmission rates, prevalence in impoverished regions, and high co-infection rates with HIV. Moreover, the serious side effects of long-term treatment that decrease patient adherence, and the emergence of multi-resistant strains of Mycobacterium tuberculosis, the causing agent of TBs, pose several challenges for its eradication. The search for a new TB treatment is necessary and urgent. Dihydroorotate dehydrogenase (DHODH) is responsible for the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway. DHODH has been considered an attractive target against infectious diseases. As a first step towards exploiting DHODH as a drug target against TB, we performed a full kinetic characterization of both bacterial MtDHODH and its human ortholog (HsDHDOH) using both substrates coenzyme Q0 (Q0) and vitamin K3 (K3). MtDHODH follows a ping-pong mechanism of catalysis and shares similar catalytic parameters with the human enzyme. Serendipitously, Q0 was found to inhibit MtDHODH (KI (Q0) = 138 ± 31 µM). To the best of our knowledge, Q0 is the first non-orotate like dihydroorotate-competitive inhibitor for class 2 DHODHs ever described. Molecular dynamics simulations along with in silico solvent mapping allowed us to successfully probe protein flexibility and correlate it with the druggability of binding sites. Together, our results provide the starting point for the design of a new generation of potent and selective inhibitors against MtDHODH.

DHODH Hot Spots: An Underexplored Source to Guide Drug Development Efforts.

BACKGROUND: Dihydroorotate dehydrogenase (DHODH) has long been recognized as an important drug target for proliferative and parasitic diseases, including compounds that exhibit trypanocidal action and broad-spectrum antiviral activity. Despite numerous and successful efforts in structural and functional characterization of DHODHs, as well as in the development of inhibitors, DHODH hot spots remain largely unmapped and underexplored. OBJECTIVE: This review describes the tools that are currently available for the identification and characterization of hot spots in protein structures and how freely available webservers can be exploited to predict DHODH hot spots. Moreover, it provides for the first time a review of the antiviral properties of DHODH inhibitors. METHODS: X-ray structures from human (HsDHODH) and Trypanosoma cruzi DHODH (TcDHODH) had their hot spots predicted by both FTMap and Fragment Hotspot Maps web servers. RESULTS: FTMap showed that hot spot occupancy in HsDHODH is correlated with the ligand efficiency (LE) of its known inhibitors, and Fragment Hotspot Maps pointed out the contribution of selected moieties to the overall LE. The conformational flexibility of the active site loop in TcDHODH was found to have a major impact on the druggability of the orotate binding site. In addition, both FTMap and Fragment Hotspot Maps servers predict a novel pocket in TcDHODH dimer interface (S6 site). CONCLUSION: This review reports how hot spots can be exploited during hit-to-lead steps, docking studies or even to improve inhibitor binding profile and by doing so using DHODH as a model, points to new drug development opportunities.

Druggable hot spots in trypanothione reductase: novel insights and opportunities for drug discovery revealed by DRUGpy.

Assessment of target druggability guided by search and characterization of hot spots is a pivotal step in early stages of drug-discovery. The raw output of FTMap provides the data to perform this task, but it relies on manual intervention to properly combine different sets of consensus sites, therefore allowing identification of hot spots and evaluation of strength, shape and distance among them. Thus, the user's previous experience on the target and the software has a direct impact on how data generated by FTMap server can be explored. DRUGpy plugin was developed to overcome this limitation. By automatically assembling and scoring all possible combinations of consensus sites, DRUGpy plugin provides FTMap users a straight-forward method to identify and characterize hot spots in protein targets. DRUGpy is available in all operating systems that support PyMOL software. DRUGpy promptly identifies and characterizes pockets that are predicted by FTMap to bind druglike molecules with high-affinity (druggable sites) or low-affinity (borderline sites) and reveals how protein conformational flexibility impacts on the target's druggability. The use of DRUGpy on the analysis of trypanothione reductases (TR), a validated drug target against trypanosomatids, showcases the usefulness of the plugin, and led to the identification of a druggable pocket in the conserved dimer interface present in this class of proteins, opening new perspectives to the design of selective inhibitors.

Synthesis and biological evaluation of thiazolidinedione derivatives with high ligand efficiency to P. aeruginosa PhzS.

The thiazolidinone ring is found in compounds that have widespan biology activity and there is mechanism-based evidence that compounds bearing this moiety inhibit P. aeruginosa PhzS (PaPzhS), a key enzyme in the biosynthesis of the virulence factor named pyocyanin. Ten novel thiazolidinone derivatives were synthesised and screened against PaPhzS, using two orthogonal assays. The biological results provided by these and 28 other compounds, whose synthesis had been described, suggest that the dihydroquinazoline ring, found in the previous hit (A- Kd = 18 µM and LE = 0.20), is not required for PaPzhS inhibition, but unsubstituted nitrogen at the thiazolidinone ring is. The molecular simplification approach, pursued in this work, afforded an optimised lead compound (13- 5-(2,4-dimethoxyphenyl)thiazolidine-2,4-dione) with 10-fold improvement in affinity (Kd= 1.68 µM) and more than 100% increase in LE (0.45), which follows the same inhibition mode as the original hit compound (competitive to NADH).Executive summaryPhzS is a key enzyme in the pyocyanin biosynthesis pathway in P. aeruginosa.Orthogonal assays (TSA and FITC) show that fragment-like thiazolidinedione derivatives bind to PaPhzS with one-digit micromolar affinity.Fragment-like thiazolidinedione derivatives bind to the cofactor (NADH) binding site in PaPhzS.The molecular simplification optimised the ligand efficiency and affinity of the lead compound.

Triazol-phenyl Antipyretic Derivatives Inhibit mPGES-1 mRNA Levels in LPS-Induced RAW 264.7 Macrophage Cells.

BACKGROUND: Microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes the terminal step of prostaglandin E2 (PGE2) production, which plays an important role in the regulation of febrile response. In our previous work, ligand-based pharmacophore models, built with mPGES-1 inhibitors, were employed to identify a novel series of compounds that reduce the febrile response in rats. OBJECTIVES: The study aimed to evaluate the mechanism of action of the most active compound (1). METHODS: For in vivo assays, rats were pretreated with the antipyretic compounds 1-8, 30 min before LPS injection. For in vitro assays, RAW 264.7 macrophage cells were incubated with the antipyretic compounds 1-8 for 1 hour before LPS stimulus. After 16 h, quantitative real-time PCR was carried out. Additionally, the PGE2 concentration in the hypothalamus was quantified by ELISA and the inhibitory effect of N-cyclopentyl-N'-[3-(3-cyclopropyl-1H-1,2,4-triazol- 5-yl)phenyl]ethanediamide (1) over human COX-2 enzymatic activity was determined with a COX Colorimetric Inhibitor Screening Assay Kit. RESULTS: Compound 1 and CAY10526 showed comparable efficacy to reduce the febrile response when injected i.v. (compound 1: 63.10%, CAY10526: 70.20%). Moreover, compound 1 significantly reduced the mPGES-1 mRNA levels, in RAW264.7 cells, under inflammatory conditions. A chemically-similar compound (8-) also significantly reduced the mRNA levels of the gene target. On the other hand, compounds 6 and 7, which are also somewhat similar to compound 1, did not significantly impact mPGES-1 mRNA levels. CONCLUSIONS: PGE2 concentration reduction in the hypothalamus, due to compound 1 central injection, is related to decreased mPGES-1 mRNA levels but not to COX-2 inhibition (IC50> 50 µM). Therefore, compound 1 is a promising lead for innovative antipyretic drug development.

Inhibition of intracellular Ca2+ mobilization and potassium channels activation are involved in the vasorelaxation induced by 7-hydroxycoumarin.

Coumarins exhibit a wide variety of biological effects, including activities in the cardiovascular system and the aim of this study was to evaluate the vascular therapeutic potential of 7-Hydroxicoumarin (7-HC). The vascular effects induced by 7-HC (0.001 µM-300 µM), were investigated by in vitro approaches using isometric tension measurements in rat superior mesenteric arteries and by in silico assays using Ligand-based analysis. Our results suggest that the vasorelaxant effect of 7-HC seems to rely on potassium channels, notably through large conductance Ca2+-activated K+ (BKCa) channels activation. In fact, 7-HC (300 µM) significantly reduced CaCl2-induced contraction as well as the reduction of intracellular calcium mobilization. However, the relaxation induced by 7-HC was independent of store-operated calcium entry (SOCE). Moreover, in silico analysis suggests that potassium channels have a common binding pocket, where 7-HC may bind and hint that its binding profile is more similar to quinine's than verapamil's. These results are compatible with the inhibition of Ca2+ release from intracellular stores, which is prompted by phenylephrine and caffeine. Taken together, these results demonstrate a therapeutic potential of 7-HC on the cardiovascular system, making it a promising lead compound for the development of drugs useful in the treatment of cardiovascular diseases.

A novel scaffold to fight Pseudomonas aeruginosa pyocyanin production: early steps to novel antivirulence drugs.

Aim: Although bacterial resistance is a growing concern worldwide, the development of antibacterial drugs has been steadily decreasing. One alternative to fight this issue relies on reducing the bacteria virulence without killing it. PhzS plays a pivotal role in pyocyanin production in Pseudomonas aeruginosa. Results: A total of 31 thiazolidinedione derivatives were evaluated as putative PhzS inhibitors, using thermo shift assays. Compounds that significantly shifted PhzS's Tm had their mode of inhibition (cofactor competitor) and affinity calculated by thermo shift assays as well. The most promising compound (E)-5-(4-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methoxy)benzylidene)thiazolidine-2,4-dione had their affinity confirmed by microscale thermophoresis (Kd = 18 µM). Cellular assays suggest this compound reduces pyocyanin production in vitro, but does not affect P. aeruginosa viability. Conclusion: The first inhibitor of PhzS is described.

Antileishmanial activity evaluation of thiazolidine-2,4-dione against Leishmania infantum and Leishmania braziliensis.

Leishmaniasis is responsible for approximately 65,000 annual deaths. Despite the mortality data, drugs available for the treatment of patients are insufficient and have moderate therapeutic efficacy in addition to serious adverse effects, which makes the development of new drugs urgent. To achieve this goal, the integration of kinetic and DSF assays against parasitic validated targets, along with phenotypic assays, can help the identification and optimization of bioactive compounds. Pteridine reductase 1 (PTR1), a validated target in Leishmania sp., is responsible for the reduction of folate and biopterin to tetrahydrofolate and tetrahydrobiopterin, respectively, both of which are essential for cell growth. In addition to the in vitro evaluation of 16 thiazolidine-2,4-dione derivatives against Leishmania major PTR1 (LmPTR1), using the differential scanning fluorimetry (ThermoFluor®), phenotypic assays were employed to evaluate the compound effect over Leishmania braziliensis (MHOM/BR/75/M2903) and Leishmania infantum (MHOM/BR/74/PP75) promastigotes viability. The ThermoFluor® results show that thiazolidine-2,4-dione derivatives have micromolar affinity to the target and equivalent activity on Leishmania cells. 2b is the most potent compound against L. infantum (EC50 = 23.45 ± 4.54 µM), whereas 2a is the most potent against L. braziliensis (EC50 = 44.16 ± 5.77 µM). This result suggests that lipophilic substituents on either-meta and/or-para positions of the benzylidene ring increase the potency against L. infantum. On the other hand, compound 2c (CE50 = 49.22 ± 7.71 µM) presented the highest selectivity index.

Calycopterin, a major flavonoid from Marcetia latifolia, modulates virulence-related traits in Pseudomonas aeruginosa.

Although bacterial resistance is a worldwide growing concern, the development of bacteriostatic and bactericidal drugs has been decreasing in the last decade. Compounds that modulate the microorganism virulence, without killing it, have been considered promising alternatives to combat bacterial infections. However, most signaling pathways that regulate virulence are complex and not completely understood. The rich chemical diversity of natural products offers a good starting point to identify key compounds that shed some light on this matter. Therefore, we investigated the role of Marcetia latifolia ethanolic extract, as well as its major constituent, calycopterin (5,4'-dihydroxy-3,6,7,8-tetramethoxylflavone), in the regulation of virulence-related phenotypes of Pseudomonas aeruginosa. Our results show that calycopterin inhibits pyocyanin production (EC50 = 32 µM), reduces motility and increases biofilm formation in a dose-dependent manner. Such biological profile suggests that calycopterin modulates targets that may act upstream the quorum sensing regulators and points to its utility as a chemical probe to further investigate P. aeruginosa transition from planktonic to sessile lifestyle.

Dual and selective inhibitors of pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Leishmania chagasi.

Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50-2.30 µM and LcDHFR Ki = 0.28-3.00 µM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 µM, selectivity index = 20).

Synthesis and Biological Evaluation of Coumarins Derivatives as Potential Inhibitors of the Production of Pseudomonas aeruginosa Virulence Factor Pyocyanin.

Antimicrobial Resistance (AMR) is a serious problem for the humans since it threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi. One way around this problem is to act on the virulence factors, produced by bacteria, which increase their infection effectiveness. In view of these facts, new coumarin derivatives were synthesized and evaluated for their anti-virulence biological activity towards Pseudomonas aeruginosa. The results suggest that coumarin derivatives with a secondary carbon at C-3 position reduces P. aeruginosa growth whereas compounds with one additional substituent have a significant effect over pyocyanin production (10k EC50 7 ± 2 µM; 10l EC50 42 ± 13 µM). Moreover, 10k reduces P. aeruginosa motility and biofilm formation, what is compatible with a quorum sensing related mechanism of action.

Virtual screening and biological evaluation of novel antipyretic compounds.

Due to the absence of safety of the antipyretics to patients with cardiovascular dysfunction, new targets to treat inflammation have been pursued. mPGES-1 is a promising target because its inhibition would not cause the side-effects related to COX inhibition. To identify novel inhibitors of mPGES-1, we developed a ligand-based pharmacophore model that differentiates true inhibitors from decoys and enlightens the structure-activity relationships for known mPGES-1 inhibitors. The model (four hydrophobic centers, two hydrogen bond acceptor and two hydrogen bond donor points) was employed to select lead-like compounds from ZINC database for in vivo evaluation. Among the 18 compounds selected, five inhibited the fever induced by LPS. The most potent compound (5-(4-fluorophenyl)-3-({6-methylimidazo[1,2-a]pyridin-2-yl}methyl)-2,3dihydro-1,3,4-oxadiazol-2-one) is active peripherally (i.v.) or centrally (i.c.v.) (82.18% and 112% reduction, respectively) and reduces (69.13%) hypothalamic PGE2 production, without significant COX-1/2 inhibition. In conclusion, our in silico approach leads to the selection of a compound that presents the chemical features to inhibit mPGES-1 and reduces fever induced by LPS. Furthermore, the in vivo and in vitro results support the hypothesis that its mechanism of action does not depend on COX inhibition. Hence, it can be considered a promising lead compound for antipyretic development, once it would not have the side-effects of COX-1/2 inhibitors.

An integrated approach towards the discovery of novel non-nucleoside Leishmania major pteridine reductase 1 inhibitors.

Despite the fact that Leishmania ssp are pteridine auxotrophs, Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) inhibitors are ineffective against Leishmania major. On the other hand Pteridine Reductase 1 (PTR1) inhibitors proved to be lethal to the parasite. Aiming at identifying hits that lie outside the chemical space of known PTR1 inhibitors, pharmacophore models that differentiate true-binders from decoys and explain the structure-activity relationships of known inhibitors were employed to virtually screen the lead-like subset of ZINC database. This approach leads to the identification of Z80393 (IC50 = 32.31 ± 1.18 µM), whose inhibition mechanism was investigated by Thermal Shift Assays. This experimental result supports a competitive mechanism and was crucial to establish the docking search space as well as select the best pose, which was then investigated by molecular dynamics studies that corroborate the hit putative binding profile towards LmPTR1. The information gathered from such studies shall be useful to design more potent non-nucleoside LmPTR1 inhibitors.

Structure-guided discovery of thiazolidine-2,4-dione derivatives as a novel class of Leishmania major pteridine reductase 1 inhibitors.

Leishmania major, as other protozoan parasites, plague human kind since pre-historic times but it remains a worldwide ailment for which the therapeutic arsenal remains scarce. Although L. major is pteridine- and purine-auxotroph, well-established folate biosynthesis inhibitors, such as methotrexate, have poor effect over the parasite survival. The lack of efficiency is related to an alternative biochemical pathway in which pteridine reductase 1 (PTR1) plays a major role. For this reason, this enzyme has been considered a promising target for anti-leishmanial drug development and several inhibitors that share the substrate scaffold have been reported. In order to design a novel class of PTR1 inhibitors, we employed the thiazolidinone ring as a bioisosteric replacement for pteridine/purine ring. Among seven novel thiazolidine-2,4-dione derivatives reported herein, 2d was identified as the most promising lead by thermal shift assays (ΔTm = 11 °C, p = 0,01). Kinetic assays reveal that 2d has IC50 = 44.67 ± 1.74 µM and shows a noncompetitive behavior. This information guided docking studies and molecular dynamics simulations (50 000 ps) that supports 2d putative binding profile (H-bonding to Ser-111 and Leu-66) and shall be useful to design more potent inhibitors.