Discovery of thiadiazole amides as potent, S1P₃-sparing agonists of sphingosine-1-phosphate 1 (S1P₁) receptor

High-throughput screening of GSK compound collection led to the discovery of a novel series of thiadiazole amides as potent and S1P(3)-sparing sphingosine-1-phosphate 1 (S1P(1)) receptor agonists. Synthesis, structure and activity relationship, selectivity, and some developability properties are described.     

Synthesis and Properties of P1, P2-, P1, P3- and P1, P4-Dinucleoside Di-, Tri- and Tetraphosphate mRNA 5′-Cap Analogues

Abstract

Chemically synthesized dinucleoside P¹, P²-di-, P¹, P³-tri- and P¹, P⁴-tetraphosphates, derivatives of 5′-linked 7-methylguanosine and guanosine were characterized with respect to their structural properties and functional effect on eukaryotic translation inhibition.     

Actions of a picomolar short-acting S1P₁ agonist in S1P₁-eGFP knock-in mice

Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment.     

Structural characterization of P1′-diversified urea-based inhibitors of glutamate carboxypeptidase II

Urea-based inhibitors of human glutamate carboxypeptidase II (GCPII) have advanced into clinical trials for imaging metastatic prostate cancer. In parallel efforts, agents with increased lipophilicity have been designed and evaluated for targeting GCPII residing within the neuraxis. Here we report the structural and computational characterization of six complexes between GCPII and P1′-diversified urea-based inhibitors that have the C-terminal glutamate replaced by more hydrophobic moieties. The X-ray structures are complemented by quantum mechanics calculations that provide a quantitative insight into the GCPII/inhibitor interactions. These data can be used for the rational design of novel glutamate-free GCPII inhibitors with tailored physicochemical properties.     

Formation of P1, P2-dinucleoside 5′-pyrophosphates under potentially prebiological conditions

Summary Organic pyrophosphates such as UppA and NAD are formed when a solution containing a nucleotide, a nucleoside 5-polyphosphate, Mg²⁺ and imidazole are allowed to dry out. We suggest that this synthesis may have occured concurrently with oligonucleotide formation.Abbreviations Im Imidazole - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - U uridine - p_nA adenosine 5-poly-phosphate containing n phosphate residues - pU uridine 5-phosphate - AppA P₁,P₂-diadenosine 5-pyrophosphate - UppA P₁-(uridine 5)-P₂-(adenosine 5)-pyrophosphate - ImpA adenosine 5-phosphorimidazolide - NMN nicotinamide mononucleotide - NAD nicotinamide-adenine dinucleotide     

Chemical Synthesis of Cap Analogues: P1,P2-Dinucleoside-5′-diphosphates

Abstract

A procedure was developed for the chemical synthesis of P¹,P²-dinucleoside-5′-diphosphates (N₁(5′)pp(5′)N₂) on a nanomolar scale Reaction conditions for activating purine-5′-monophosphates (pA, pG, and pm⁷G) by 1,1′-carbonyldiimidazole were studied and optimized in respect to solvents and amount of activating reagent used. Various dinucleoside-5′-diphosphates were synthesized in 62-98% yield by incubating activated and non-activated purine-5′-monophosphates. Two unexpected by-products were formed by competition reactions: the imidazolidate of the non-activated nucleotide and the corresponding symmetrically substituted dinucleoside-5′-diphosphate. A mechanism is proposed to explain the observed side reactions.     

Exploration of cathepsin S inhibitors characterized by a triazole P1–P2 amide replacement

This paper details exploration of a class of triazole-based cathepsin S inhibitors originally reported by Ellman and co-workers. SAR studies involving modifications across the whole inhibitor provide a perspective on the strengths and weaknesses of this class of inhibitors. In addition, we put the unique characteristics of this class of compounds into perspective with other classes of cathepsin S inhibitors.     

Characteristics and application of S1–P1 nucleases in biotechnology and medicine

3′–nucleases/nucleotidases of the S1–P1 family (EC 3.1.30.1) are single–strand–specific or non-specific zinc–dependent phosphoesterases present in plants, fungi, protozoan parasites, and in some bacteria. They participate in a wide variety of biological processes and their current biotechnological applications rely on their single–strand preference, nucleotide non-specificity, a broad range of catalytic conditions and high stability. We summarize the present and potential utilization of these enzymes in biotechnology and medicine in the context of their biochemical and structure–function properties. Explanation of unanswered questions for bacterial and trypanosomatid representatives could facilitate development of emerging applications in medicine.     

Characterization of bacteriophage P1 library containing inserts of Drosophila DNA of 75–100 kilobase pairs

A multiple-hit bacteriophage P1 library containing DNA fragments from Drosophila melanogaster in the size range 75–100 kb was created and subjected to a preliminary evaluation for completeness, randomness, fidelity, and clone stability. This P1 library presently contains 3840 individual clones, or approximately two genome equivalents. The library was screened with a small set of unique-sequence test probes, and clones containing the sequences have been recovered. In situ hybridization with salivary gland chromosomes indicates that the clones originate from the site of the probe sequences in the genome, and filter hybridization of restriction digests suggests that the clones are not rearranged in comparison with the genomic sequences. Approximately 1.7% of the clones contain sequences that hybridize with ribosomal DNA. A small subset of these clones was tested for stability by examination of restriction fragments produced after repeated subculturing, and no evidence for instability was found. The P1 cloning system has general utility in molecular genetics and may provide an important intermediate level of resolution in physical mapping of the Drosophila genome.by W. Hennig     

Synthesis and activity of quinolinylmethyl P1′ α-sulfone piperidine hydroxamate inhibitors of TACE

A series of α-sulfone piperidine hydroxamate TACE inhibitors 11a–n bearing a quinolinyl methyl P1′ group was prepared, and their activity was compared to analogous α- and β-sulfone piperidine hydroxamates with a butynyloxy P1′ group. The quinolinyl methyl P1′ group affords increased inhibitory enzyme activity relative to the corresponding butynyloxy P1′ analogs in the α-sulfone piperidine hydroxamate series, and greater selectivity than the corresponding butynyloxy P1′ analogs in the β-sulfone piperidine hydroxamate series.     

Gαq-mediated plasma membrane translocation of sphingosine kinase-1 and cross-activation of S1P receptors

Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of G_q-coupled receptors induced a profound, rapid (half-life 3–5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein α-subunits specifically of the G_q family. Classical G_q signalling pathways, or phosphorylation at Ser²²⁵, phospholipase D and Ca²⁺/calmodulin were not involved in M₃ receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of G_q-coupled receptors, linking them via cross-activation of S1P receptors to G_i and G_12/13 signalling pathways.     

Hydroxyethylamine-based inhibitors of BACE1: P1–P3 macrocyclization can improve potency,selectivity, and cell activity

We describe a systematic study of how macrocyclization in the P₁–P₃ region of hydroxyethylamine-based inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) modulates in vitro activity. This study reveals that in a number of instances macrocyclization of bis-terminal dienes leads to improved potency toward BACE1 and selectivity against cathepsin D (CatD), as well as greater amyloid β-peptide (Aβ)-lowering activity in HEK293T cells stably expressing APP_SW. However, for several closely related analogs the benefits of macrocyclization are attenuated by the effects of other structural features in different regions of the molecules. X-ray crystal structures of three of these novel macrocyclic inhibitors bound to BACE1 revealed their binding conformations and interactions with the enzyme.     

SAR analysis of innovative selective small molecule antagonists of sphingosine-1-phosphate 4 (S1P₄) receptor

Recent evidence suggests an innovative application of chemical modulators targeting the S1P(4) receptor as novel mechanism-based drugs for the treatment of influenza virus infection. Modulation of the S1P(4) receptor may also represent an alternative therapeutic approach for clinical conditions where reactive thrombocytosis is an undesired effect or increased megakaryopoiesis is required. With the exception of our recent research program disclosure, we are not aware of any selective S1P(4) antagonists reported in the literature to date. Herein, we describe complementary structure-activity relationships (SAR) of the high-throughput screening (HTS)-derived hit 5-(2,5-dichlorophenyl)-N-(2,6-dimethylphenyl)furan-2-carboxamide and its 2,5-dimethylphenyl analog. Systematic structural modifications of the furan ring showed that both steric and electronic factors in this region have a significant impact on the potency. The furan moiety was successfully replaced with a thiophene or phenyl ring maintaining potency in the low nanomolar range and high selectivity against the other S1P receptor subtypes. By expanding the molecular diversity within the hit-derived class, our SAR study provides innovative small molecule potent and selective S1P(4) antagonists suitable for in vivo pharmacological validation of the target receptor.     

Antiplatelet Activity,P2Y1 and P2Y12 Inhibition,and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate

Background

Diadenosine tetraphosphate (Ap₄A), a constituent of platelet dense granules, and its P¹,P⁴-dithio and/or P²,P³-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y₁ and P2Y₁₂. The most active of those analogs, diadenosine 5′,5″″-P¹,P⁴-dithio-P²,P³-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.

Objective

To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y₁ and P2Y₁₂ receptor antagonism, and their metabolism in human plasma.

Methods

We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y₁-mediated changes in cytosolic Ca²⁺, P2Y₁₂-mediated changes in VASP phosphorylation, and metabolism in human plasma.

Results

The inhibition of ADP-induced human platelet aggregation and human platelet P2Y₁₂ receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P¹- and P⁴-phosphorothioate groups, the S_PS_P diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the R_PR_P diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of S_PR_P diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P²,P³-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the S_PS_P and the R_PR_P diastereomers.

Conclusions

The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y₁ and P2Y₁₂ receptors.     

S1P₂ receptor regulation of sphingosine-1-phosphate effects on conventional outflow physiology

Elevated intraocular pressure is the main risk factor in primary open-angle glaucoma, involving an increased resistance to aqueous humor outflow in the juxtacanalicular region of the conventional outflow pathway which includes the trabecular meshwork (TM) and the inner wall of Schlemm's canal (SC). Previously, sphingosine-1-phosphate (S1P) was shown to decrease outflow facility in porcine and human eyes, thus increasing outflow resistance and intraocular pressure. Owing to S1P's known effect of increasing barrier function in endothelial cells and the robust expression of the S1P? receptor on the inner wall of SC, we hypothesized that S1P? receptor activation promotes junction formation and decreases outflow facility. The effects of subtype-specific S1P receptor compounds were tested in human and porcine whole-eye perfusions and human primary cultures of SC and TM cells to determine the receptor responsible for S1P effects on outflow resistance. The S1P?-specific agonist SEW2871 failed to both mimic S1P effects in paired human eye perfusions, as well as increase myosin light chain (MLC) phosphorylation in cell culture, a prominent outcome in S1P-treated SC and TM cells. In contrast, the S1P? antagonist JTE-013, but not the S1P? or S1P?,? antagonists, blocked the S1P-promoted increase in MLC phosphorylation. Moreover, JTE-013 prevented S1P-induced decrease in outflow facility in perfused human eyes (P < 0.05, n = 6 pairs). Similarly, porcine eyes perfused with JTE-013 + S1P did not differ from eyes with JTE-013 alone (P = 0.53, n = 3). These results demonstrate that S1P? , and not S1P? or S1P?, receptor activation increases conventional outflow resistance and is a potential target to regulate intraocular pressure.     

Involvement of Cytochrome P450 1A1 and Glutathione S-Transferase P1 Polymorphisms and Promoter Hypermethylation in the Progression of Anti-Tuberculosis Drug-Induced Liver Injury: A Case–Control Study

Background

Anti-tuberculosis (anti-TB) drug-induced liver injury (ADLI) is one of the most common adverse effects associated with TB treatment. Cytochrome P450 (CYP) 1A1 and glutathione S-transferase (GST) P1 are important phase I/II metabolizing enzymes involved in drug metabolism and detoxification. Genetic polymorphism and CpG island methylation have been reported as factors influencing the expression of CYP1A1 and GSTP1.

Objective

This study aimed to determine the potential relationships of CYP1A1 and GSTP1 polymorphisms and CpG island methylation with ADLI risk.

Design

This was a population-based one-to-one matched case–control study.

Setting

The subjects were patients with TB receiving treatment in China from December 2010 to June 2013.

Patients

In total, 127 patients with TB and ADLI (case group) and 127 patients with TB but without liver injury (control group) were included in this study. Subjects were matched in terms of sex, age, and therapeutic regimen.

Methods

The general condition of each patient was assessed using questionnaires. The CYP1A1 MspI and GSTP1 Ile105Val polymorphisms as well as methylation status were detected by polymerase chain reaction (PCR)–restriction fragment length polymorphism and the methylation-specific PCR method.

Results

We found no significant difference in GSTP1 and CYP1A1 genotypes between the two groups, probably because the sample size was not large enough; however, patients with ADLI had significantly higher GSTP1 and CYP1A1 promoter methylation rates than control subjects [odds ratio (OR) = 2.467 and 2.000, respectively]. After adjusting for drinking, which significantly differed between the groups as per univariate analysis, we found that hypermethylation of GSTP1 and CYP1A1 promoters was associated with ADLI (OR = 2.645 and 2.090, respectively).

Conclusion

Hypermethylation of CpG islands of GSTP1 and CYP1A1 promoters may thus play important roles in the development of ADLI and provide evidence of being used as novel markers for ADLI risk prediction.     

Kinetics of inhibition of ribonuclease P activity by peptidyltransferase inhibitors – Effect of antibiotics on RNase P

A cell-free system derived from Dictyostelium discoideum has been used to study the kinetics of inhibition of RNase P by puromycin, amicetin and blasticidin S. Detailed kinetic analysis showed that the type of inhibition of RNase P activity by puromycin is simple competitive, whereas the type of inhibition by amicetin and blasticidin S is simple non-competitive. On the basis of K_i values amicetin is stronger inhibitor than puromycin and blasticidin S.     

Binding of organic solvent molecules influences the P1′−P2′ stereo-and sequence-specificity of α-chymotrypsin in kinetically controlled peptide synthesis

In kinetically controlled peptide synthesis catalyzed by -chymotrypsin (EC 3.4.21.1, CT) the P₁-specificity and stereospecificity was found to increase for hydrophobic and decrease for positively charged nucleophiles in systems with hydrophobic organic solvent molecules. This was observed in the following systems: (i) buffer saturated with organic solvent molecules; (ii) the enzyme adsorbed on a hydrophobic adsorbent (Phenylbutylamine-Eupergit). These results show that organic molecules bound to the enzyme surface perturb the P₁-S₁ interactions and change the catalytic properties of the enzyme.