Inhibition of CTP synthase from Escherichia coli by xanthines and uric acids

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a recognized target for the development of anticancer, antiviral, and antiprotozoal agents. Xanthine and related compounds inhibit CTPS activity (IC₅₀ = 0.16–0.58 mM). The presence of an 8-oxo function (i.e., uric acids) enhances inhibition (IC₅₀ = 0.060–0.121 mM). An intact purine ring with anionic character favors inhibition. In general, methylation of the purine does not significantly affect inhibition.     

Inhibition of glutamate racemase by substrate–product analogues

d-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bacterial cell wall. Glutamate racemase catalyzes the reversible formation of d-glutamate from l-glutamate and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate–product analogue (R,S)-1-hydroxy-1-oxo-4-amino-4-carboxyphosphorinane is a modest, partial noncompetitive inhibitor of glutamate racemase from Fusobacterium nucleatum (FnGR), a pathogen responsible, in part, for periodontal disease and colorectal cancer (K_i = 3.1 ± 0.6 mM, cf. K_m = 1.41 ± 0.06 mM). The cyclic substrate–product analogue (R,S)-4-amino-4-carboxy-1,1-dioxotetrahydro-thiopyran was a weak inhibitor, giving only ∼30% inhibition at a concentration of 40 mM. The related cyclic substrate–product analogue 1,1-dioxo-tetrahydrothiopyran-4-one was a cooperative mixed-type inhibitor of FnGR (K_i = 18.4 ± 1.2 mM), while linear analogues were only weak inhibitors of the enzyme. For glutamate racemase, mimicking the structure of both enantiomeric substrates (substrate–product analogues) serves as a useful design strategy for developing inhibitors. The new cyclic compounds developed in the present study may serve as potential lead compounds for further development.     

The synthesis of 4,7-disubstituted-2H-benzo[b][1,4]-oxazin-3(4H)-ones using Smiles rearrangement and their in vitro evaluation as platelet aggregation inhibitors

A series of novel benzo[b][1,4]oxazin-3(4H)-one derivatives were synthesized as platelet aggregation inhibitors for structure–activity relationships (SAR) analysis. The synthetic pattern, involved Smiles rearrangement for the preparation of benzoxazine, was proven to be more efficient than the conventional methods. Biological evaluation demonstrated that among all the synthesized compounds, compound 9u (IC₅₀ = 9.20 μM) exhibited the most potent inhibition activity compared with aspirin, the positive control (IC₅₀ = 7.07 μM). Molecular docking revealed that these set of compounds could be the GPIIb/IIIa antagonist for that they could be situated in the binding site of GPIIb/IIIa receptor quite well.     

Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity

The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A_2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A_2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A_2A adenosine receptor ligand with K_i = 0.06 μM. Similarly potent and highly A_2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs.     

Mechanism-based inhibitors of serine proteases with high selectivity through optimization of S′ subsite binding

A series of mechanism-based inhibitors designed to interact with the S′ subsites of serine proteases was synthesized and their inhibitory activity toward the closely-related serine proteases human neutrophil elastase (HNE) and proteinase 3 (PR 3) was investigated. The compounds were found to be time-dependent inhibitors of HNE and were devoid of any inhibitory activity toward PR 3. The results suggest that highly selective inhibitors of serine proteases whose primary substrate specificity and active sites are similar can be identified by exploiting differences in their S′ subsites. The best inhibitor (compound 16) had a k_inact/K_I value of 4580 M^?1 s^?1.     

In vitro characterization of CYP102G4 from Streptomyces cattleya: A self-sufficient P450 naturally producing indigo

Self-sufficient CYP102As possess outstanding hydroxylating activity to fatty acids such as myristic acid. Other CYP102 subfamily members share substrate specificity of CYP102As, but, occasionally, unusual characteristics of its own subfamily have been found. In this study, only one self-sufficient cytochrome P450 from Streptomyces cattleya was renamed from CYP102A_scat to CYP102G4, purified and characterized. UV–Vis spectrometry pattern, FAD/FMN analysis, and protein sequence comparison among CYP102s have shown that CYP102 from Streptomyces cattleya belongs to CYP102G subfamily. It showed hydroxylation activity toward fatty acids generating ω-1, ω-2, and ω-3-hydroxyfatty acids, which is similar to the general substrate specificity of CYP102 family. Unexpectedly, however, expression of CYP102G4 showed indigo production in LB medium batch flask culture, and high catalytic activity (k_cat/K_m) for indole was measured as 6.14 ± 0.10 min^− 1 mM^− 1. Besides indole, CYP102G4 was able to hydroxylate aromatic compounds such as flavone, benzophenone, and chloroindoles. Homology model has shown such ability to accept aromatic compounds is due to its bigger active site cavity. Unlike other CYP102s, CYP102G4 did not have biased cofactor dependency, which was possibly determined by difference in NAD(P)H binding residues (Ala984, Val990, and Tyr1064) compared to CYP102A1 (Arg966, Lys972 and Trp1046). Overall, a self-sufficient CYP within CYP102G subfamily was characterized using purified enzymes, which appears to possess unique properties such as an only prokaryotic CYP naturally producing indigo.     

Coumarin-thiazole and -oxadiazole derivatives: Synthesis,bioactivity and docking studies for aldose/aldehyde reductase inhibitors

In continuation of our previous efforts directed towards the development of potent and selective inhibitors of aldose reductase (ALR2), and to control the diabetes mellitus (DM), a chronic metabolic disease, we synthesized novel coumarin-thiazole 6(a–o) and coumarin-oxadiazole 11(a–h) hybrids and screened for their inhibitory activity against aldose reductase (ALR2), for the selectivity against aldehyde reductase (ALR1). Compounds were also screened against ALR1. Among the newly designed compounds, 6c, 11d, and 11g were selective inhibitors of ALR2. Whereas, (E)-3-(2-(2-(2-bromobenzylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one 6c yielded the lowest IC₅₀ value of 0.16 ± 0.06 μM for ALR2. Moreover, compounds (E)-3-(2-(2-benzylidenehydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6a; IC₅₀ = 2.94 ± 1.23 μM for ARL1 and 0.12 ± 0.05 μM for ARL2) and (E)-3-(2-(2-(1-(4-bromophenyl)ethylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6e; IC₅₀ = 1.71 ± 0.01 μM for ARL1 and 0.11 ± 0.001 μM for ARL2) were confirmed as dual inhibitors. Furthermore, compounds 6i, 6k, 6m, and 11b were found to be selective inhibitors for ALR1, among which (E)-3-(2-(2-((2-amino-4-chlorophenyl)(phenyl)methylene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (6m) was most potent (IC₅₀ = 0.459 ± 0.001 μM). Docking studies performed using X-ray structures of ALR1 and ALR2 with the given synthesized inhibitors showed that coumarinyl thiazole series lacks the carboxylate function that could interact with the anionic binding site being a common ALR1/ALR2 inhibitors trait. Molecular docking study with dual inhibitor 6e also suggested plausible binding modes for the ALR1 and ALR2 enzymes. Hence, the results of this study revealed that coumarinyl thiazole and oxadiazole derivatives could act as potential ALR1/ALR2 inhibitors.     

Crystal structure and molecular dynamics studies of human purine nucleoside phosphorylase complexed with 7-deazaguanine

In humans, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. HsPNP is a target for inhibitor development aiming at T-cell immune response modulation. Here we report the crystal structure of HsPNP in complex with 7-deazaguanine (HsPNP:7DG) at 2.75 Å. Molecular dynamics simulations were employed to assess the structural features of HsPNP in both free form and in complex with 7DG. Our results show that some regions, responsible for entrance and exit of substrate, present a conformational variability, which is dissected by dynamics simulation analysis. Enzymatic assays were also carried out and revealed that 7-deazaguanine presents a lower inhibitory activity against HsPNP (K_i = 200 μM). The present structure may be employed in both structure-based design of PNP inhibitors and in development of specific empirical scoring functions.     

Investigations into specificity of azepinomycin for inhibition of guanase: Discrimination between the natural heterocyclic inhibitor and its synthetic nucleoside analogues

In our long and broad program to explore structure–activity relationships of the natural product azepinomycin and its analogues for inhibition of guanase, an important enzyme of purine salvage pathway of nucleic acid metabolism, it became necessary to investigate if the nucleoside analogues of the heterocycle azepinomycin, which are likely to be formed in vivo, would be more or less potent than the parent heterocycle. To this end, we have resynthesized both azepinomycin (1) and its two diastereomeric nucleoside analogues (2 and 3), employing a modified, more efficient procedure, and have biochemically screened all three compounds against a mammalian guanase. Our results indicate that the natural product is at least 200 times more potent toward inhibition of guanase as compared with its nucleoside analogues, with the observed K_i of azepinomycin (1) against the rabbit liver guanase = 2.5 (±0.6) × 10^?6 M, while K_i of Compound 2 = 1.19 (±0.02) × 10^?4 M and that of Compound 3 = 1.29 (±0.03) × 10^?4 M. It is also to be noted that while IC₅₀ value of azepinomycin against guanase in cell culture has long been reported, no inhibition studies nor K_i against a pure mammalian enzyme have ever been documented. In addition, we have, for the first time, determined the absolute stereochemistry of the 6-OH group of 2 and 3 using conformational analysis coupled with 2-D ¹H NMR NOESY     

Bacterial Peptide deformylase inhibition of cyano substituted biaryl analogs: Synthesis,in vitro biological evaluation,molecular docking study and in silico ADME prediction

Herein, we report the synthesis and screening of cyano substituted biaryl analogs 5(a–m) as Peptide deformylase (PDF) enzyme inhibitors. The compounds 5a (IC₅₀ value = 13.16 μM), 5d (IC₅₀ value = 15.66 μM) and 5j (IC₅₀ value = 19.16 μM) had shown good PDF inhibition activity. The compounds 5a (MIC range = 11.00–15.83 μg/mL), 5b (MIC range = 23.75–28.50 μg/mL) and 5j (MIC range = 7.66–16.91 μg/mL) had also shown potent antibacterial activity when compared with ciprofloxacin (MIC range = 25–50 μg/mL). Thus, the active derivatives were not only potent PDF inhibitors but also efficient antibacterial agents. In order to gain more insight on the binding mode of the compounds with PDF, the synthesized compounds 5(a–m) were docked against PDF enzyme of Escherichia coli and compounds exhibited good binding properties. In silico ADME properties of synthesized compounds were also analyzed and showed potential to develop as good oral drug candidates.     

Development of fluorinated methoxylated chalcones as selective monoamine oxidase-B inhibitors: Synthesis,biochemistry and molecular docking studies

A series of methoxylated chalcones with fluoro and trifluoromethyl derivatives were synthesized and investigated for their ability to inhibit human monoamine oxidase A and B. The chemical structures of the compounds have been characterized by means of their ¹H NMR, ¹³C NMR, Mass spectroscopic datas and elemental analysis. The results demonstrate that these compounds are reversible and selective MAO-B inhibitors with a competitive mode of inhibition. The most potent compound (2E)-1-(4-methoxyphenyl)-3-[4-(trifluoromethyl)phenyl] prop-2-en-1-one showed the best activity and higher selectivity towards hMAO-B with K_i and SI values of 0.22 ± 0.01 μM and 0.05 comparable to that standard drug, Selegiline K_i and SI values were found as 0.33 ± 0.03 μM and 0.04, respectively. Molecular docking studies were carried out to further explain the in vitro results of the new compounds, and to identify the hypothetical binding mode for the compounds inside the inhibitor binding cavity of hMAO-B.     

Characterization and kinetic properties of the purified Trematosphaeria mangrovei laccase enzyme

The properties of Trematosphaeria mangrovei laccase enzyme purified on Sephadex G-100 column were investigated. SDS–PAGE of the purified laccase enzyme showed a single band at 48 kDa. The pure laccase reached its maximal activity at temperature 65 °C, pH 4.0 with K_m equal 1.4 mM and V_max equal 184.84 U/mg protein. The substrate specificity of the purified laccase was greatly influenced by the nature and position of the substituted groups in the phenolic ring. The pure laccase was tested with some metal ions and inhibitors, FeSO₄ completely inhibited laccase enzyme and also highly affected by (NaN₃) at a concentration of 1 mM. Amino acid composition of the pure enzyme was also determined. Carbohydrate content of purified laccase enzyme was 23% of the enzyme sample. The UV absorption spectra of the purified laccase enzyme showed a single peak at 260–280 nm.     

Discovery,characterization and comparison of inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicases

Antibacterial compounds with new mechanisms of action are needed for effective therapy against drug-resistant pathogens in the clinic and in biodefense. Screens for inhibitors of the essential replicative helicases of Bacillus anthracis and Staphylococcus aureus yielded 18 confirmed hits (IC₅₀ ? 25 μM). Several (5 of 18) of the inhibitors were also shown to inhibit DNA replication in permeabilized polA-deficient B. anthracis cells. One of the most potent inhibitors also displayed antibacterial activity (MIC ～5 μg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial versus mammalian cells (CC₅₀/MIC > 16) suitable for further optimization. This compound shares the bicyclic ring of the clinically proven aminocoumarin scaffold, but is not a gyrase inhibitor. It exhibits a mixed mode of helicase inhibition including a component of competitive inhibition with the DNA substrate (K_i = 8 μM) and is rapidly bactericidal at 4 × MIC.     

Synthesis,in vitro evaluation and molecular docking studies of novel amide linked triazolyl glycoconjugates as new inhibitors of α-glucosidase

A series of N-substituted amide linked triazolyl β-d-glucopyranoside derivatives (4a-l) were synthesized and their in vitro inhibitory activity against yeast α-glucosidase enzyme [EC.3.2.1.20] was assessed. Compounds 4e (IC₅₀ = 156.06 μM), 4f (IC₅₀ = 147.94 μM), 4k (IC₅₀ = 127.71 μM) and 4l (IC₅₀ = 121.33 μM) were identified as the most potent inhibitors for α-glucosidase as compared to acarbose (IC₅₀ = 130.98 μM) under the same in vitro experimental conditions. Kinetic study showed that both 4e and 4f inhibit the enzyme in a competitive manner with p-nitrophenyl α-d-glucopyranoside as substrate. Molecular docking studies of 4e, 4f, 4k and 4l were also carried out using homology model of α-glucosidase to find out the binding modes responsible for the inhibitory activity. This study revealed that the binding affinity of compounds 4e, 4f, 4k and 4l for α-glucosidase were −8.2, −8.6, −8.3 and −8.5 kcal/mol respectively, compared to that of acarbose (−8.9 kcal/mol). The results suggest that the N-substituted amide linked triazole glycoconjugates can reasonably mimic the substrates for the yeast α-glucosidase.     

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing two urea groups has been developed. Inhibition potency of the described compounds ranges from 2.0 μM to 0.4 nM. 1,6-(Hexamethylene)bis[(adamant-1-yl)urea] (3b) was found to be a potent slow tight binding inhibitor (IC₅₀ = 0.5 nM) with a strong binding to sEH (K_i = 3.1 nM) and a moderately long residence time on the enzyme (k_off = 1.05 × 10⁻³ s⁻¹; t_1/2 = 11 min).     

Structure-based design of HSPA5 inhibitors: From peptide to small molecule inhibitors

We identified nine small-molecule hit compounds of Heat shock 70 kDa protein 5 (HSPA5) from cascade in silico screening based on the binding modes of the tetrapeptides derived from the peptide substrate or inhibitors of Escherichia coli HSP70. Two compounds exhibit promising inhibition activities from cancer cell viability and tumor inhibition assays. The binding modes of the hit compounds provide a platform for development of selective small molecule inhibitors of HSPA5.     

Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX

BackgroundHuman carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding.MethodsThe observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry.ResultsThe pK_a of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was − 24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM.ConclusionsThe intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship.General significanceIt is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations.     

24-Methylenecyclopropane steroidal inhibitors: A Trojan horse in ergosterol biosynthesis that prevents growth of Trypanosoma brucei

A new class of steroidal therapeutics based on phylogenetic-guided design of covalent inhibitors that target parasite-specific enzymes of ergosterol biosynthesis is shown to prevent growth of the protozoan-Trypanosoma brucei, responsible for sleeping sickness. In the presence of approximately 15 ± 5 μM 26,27-dehydrolanosterol, T. brucei procyclic or blood stream form growth is inhibited by 50%. This compound is actively converted by the parasite to an acceptable substrate of sterol C24-methyl transferase (SMT) that upon position-specific side chain methylation at C26 inactivates the enzyme. Treated cells show dose-dependent depletion of ergosterol and other 24β-methyl sterols with no accumulation of intermediates in contradistinction to profiles typical of tight binding inhibitor treatments to azoles showing loss of ergosterol accompanied by accumulation of toxic 14-methyl sterols. HEK cells accumulate 26,27-dehydrolanosterol without effect on cholesterol biosynthesis. During exposure of cloned TbSMT to 26,27-dehydrozymosterol, the enzyme is gradually inactivated (k_cat/k_inact = 0.13 min^− 1/0.08 min^− 1; partition ratio of 1.6) while 26,27-dehydrolanosterol binds nonproductively. GC–MS analysis of the turnover product and bound intermediate released as a C26-methylated diol (C3-OH and C24-OH) confirmed substrate recognition and covalent binding to TbSMT. This study has potential implications for design of a novel class of chemotherapeutic leads functioning as mechanism-based inhibitors of ergosterol biosynthesis to treat neglected tropical diseases.     

The discovery of novel and selective fatty acid binding protein 4 inhibitors by virtual screening and biological evaluation

Adipocyte fatty acid binding protein (AFABP, FABP4) has been proven to be a potential therapeutic target for diabetes, atherosclerosis and inflammation-related diseases. In this study, a series of new scaffolds of small molecule inhibitors of FABP4 were identified by virtual screening and were validated by a bioassay. Fifty selected compounds were tested, which led to the discovery of seven hits. Structural similarity-based searches were then performed based on the hits and led to the identification of one high affinity compound 33b (K_i = 0.29 ± 0.07 μM, ΔT_m = 8.5 °C). This compound’s effective blockade of inflammatory response was further validated by its ability to suppress pro-inflammatory cytokines induced by lipopolysaccharide (LPS) stimulation. Molecular dynamics simulation (MD) and mutagenesis studies validated key residues for its inhibitory potency and thus provide an important clue for the further development of drugs.     

Design,synthesis and biological evaluation of hydroxy substituted amino chalcone compounds for antityrosinase activity in B16 cells

A series of hydroxy substituted amino chalcone compounds have been synthesized. These compounds were then evaluated for their inhibitory activities on tyrosinase and melanogenesis in murine B16F10 melanoma cell lines. The structures of the compounds synthesized were confirmed by ¹H NMR, ¹³C NMR, FTIR and HRMS. Two novel amino chalcone compounds exhibited higher tyrosinase inhibitory activities (IC₅₀ values of 9.75 μM and 7.82 μM respectively) than the control kojic acid (IC₅₀: 22.83 μM). Kinetic studies revealed them to act as competitive tyrosinase inhibitors with their K_i values of 4.82 μM and 1.89 μM respectively. Both the compounds inhibited melanin production and tyrosinase activity in B16 cells. Docking results confirm that the active inhibitors strongly interact with mushroom tyrosinase residues. This study suggests that the depigmenting effect of novel amino chalcone compounds might be attributable to inhibition of tyrosinase activity, suggesting amino chalcones to be a promising candidate for use as depigmentation agents or as anti-browning food additives.