Design and synthesis of several small-size HTLV-I protease inhibitors with different hydrophilicity profiles

The human T cell leukemia/lymphotropic virus type 1 (HTLV-I) is clinically associated with adult T cell leukemia/lymphoma, HTLV-I associated myelopathy/tropical spastic paraparesis, and a number of other chronic inflammatory diseases. To stop the replication of the virus, we developed highly potent tetrapeptidic HTLV-I protease inhibitors. In a recent X-ray crystallography study, several of our inhibitors could not form co-crystal complexes with the protease due to their high hydrophobicity. In the current study, we designed, synthesized and evaluated the HTLV-I protease inhibition potency of compounds with hydrophilic end-capping moieties with the aim of improving pharmaceutic and pharmacokinetic properties.     

Locking the two ends of tetrapeptidic HTLV-I protease inhibitors inside the enzyme

Adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy are only some of the more common end results of an infection with a human T-cell leukemia virus type 1 (HTLV-I). Expanding from our previous reports, we synthesized all different permutations of tetrapeptidic HTLV-I protease inhibitors using at least eight P(3)-cap and five P(1)(')-cap moieties. The inhibitors exhibited over 97% inhibition against HIV-1 protease and a wide range of inhibitory activity against HTLV-I protease.     

Truncation and non-natural amino acid substitution studies on HTLV-I protease hexapeptidic inhibitors

The culprit behind adult T-cell leukemia, myelopathy/tropical paraparesis, and a plethora of inflammatory diseases is the human T-cell leukemia virus type 1 (HTLV-I). We recently unveiled a potent hexapeptidic HTLV-I protease inhibitor, KNI-10166, composed mostly of natural amino acid residues. Herein, we report the derivation of potent tetrapeptidic inhibitor KNI-10516, possessing only non-natural amino acid residues.     

Development of substrate analogue inhibitors for the human airway trypsin-like protease HAT

A series of substrate analogue inhibitors of the serine protease HAT, containing a 4-amidinobenzylamide moiety as the P1 residue, was prepared. The most potent compounds possess a basic amino acid in the d-configuration as P3 residue. Whereas inhibitor 4 (K_i 13 nM) containing proline as the P2 residue completely lacks selectivity, incorporation of norvaline leads to a potent inhibitor (15, K_i 15 nM) with improved selectivity for HAT in comparison to the coagulation proteases thrombin and factor Xa or the fibrinolytic plasmin. Selected inhibitors were able to suppress influenza virus replication in a HAT-expressing MDCK cell model.     

Development of [Ile40]HTLV‐I protease inhibition assay using novel fluorogenic and chromogenic substrate

HTLV‐I is a debilitating and/or lethal retrovirus that causes HTLV‐I‐associated myelopathy/tropical spastic paraparesis, adult T‐cell leukemia and several inflammatory diseases. HTLV‐I protease is an aspartic retropepsin involved in HTLV‐I replication and its inhibition could treatHTLV‐I infection. A recombinant L40I mutant HTLV‐I protease was designed and obtained from Escherichia coli, self‐processingand purification by ion‐exchange chromatography. The protease was refolded by a one‐step dialysis and recovered activity. The cleavage efficiency of the [Ile⁴⁰]HTLV‐I protease was at least 300 times higher for a fluorescent substratethan that of our previously reported recombinant His‐tagged non‐mutated HTLV‐I protease. In addition, we designed and synthesized a substrate containing a highly fluorescent Mca moiety in the fragment before the scissile bond, and a chromogenic p‐nitrophenylalanine moiety after the scissile bond that greatly amplified spectrometry detection and improved the HTLV‐I protease inhibition potency assay. The HTLV‐I protease inhibition assay with the [Ile⁴⁰]HTLV‐I protease and fluorogenic substrate requires distinctively less protease, substrate, inhibitor and assay time than our previous methods. This means our new assay is more cost‐effective and more time‐efficient while being reproducible and less labor‐intensive. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

An inhibition model of BPTI to unlinked dengue virus NS2B-NS3 protease

One approach to treating the dengue virus infection is to inhibit its NS2B-NS3 protease that plays a vital role in virus maturation. However, the lack of structural information on the active conformation of the protease hindered related drug design. With a co-expression system, we obtained the active two-component protease in its unlinked form. BPTI shows strong competitive inhibitory activity (K_i = 6.5 nM) against this unlinked protease, which adopts a closed conformation. Based on the biochemical and NMR perturbation information, an inhibition model of BPTI to NS2B-NS3 protease is proposed.     

A small compound targeting the interaction between nonstructural proteins 2B and 3 inhibits dengue virus replication

The non-structural protein NS2B/NS3 serine-protease complex of the dengue virus (DENV) is required for the maturation of the viral polyprotein. Dissociation of the NS2B cofactor from NS3 diminishes the enzymatic activity of the complex. In this study, we identified a small molecule inhibitor that interferes with the interaction between NS2B and NS3 using structure-based screening and a cell-based viral replication assay. A library containing 661,417 small compounds derived from the Molecular Operating Environment lead-like database was docked to the NS2B/NS3 structural model. Thirty-nine compounds with high scores were tested in a secondary screening using a cell-based viral replication assay. SK-12 was found to inhibit replication of all DENV serotypes (EC₅₀ = 0.74–4.92 μM). In silico studies predicted that SK-12 pre-occupies the NS2B-binding site of NS3. Steady-state kinetics using a fluorogenic short peptide substrate demonstrated that SK-12 is a noncompetitive inhibitor against the NS2B/NS3 protease. Inhibition to Japanese encephalitis virus by SK-12 was relatively weak (EC₅₀ = 29.81 μM), and this lower sensitivity was due to difference in amino acid at position 27 of NS3. SK-12 is the promising small-molecule inhibitor that targets the interaction between NS2B and NS3.     

A novel in vivo regulatory role of P-glycoprotein in alloimmunity

P-glycoprotein (P-gp) is required for adaptive immunity through defined functions in T cell activation and antigen presenting cell (APC) maturation. The potential role of P-gp as an in vivo regulator of alloimmunity is currently unknown. Here we show that P-gp blockade prolongs graft survival in a murine heterotopic cardiac allotransplantation model through in vivo inhibition of the T helper 1 (Th1) cytokine IFN-γ and the Th2 product IL-4, and via downregulation of the APC-expressed positive costimulatory molecule CD80. In vitro, the P-gp antagonist PSC833, a non-calcineurin-inhibitory cyclosporine A analogue, specifically inhibited cellular efflux of the P-gp substrate rhodamine-123 in wild-type CD3⁺ T cells and MHC class II⁺ APCs but not their P-gp knockout counterparts that lacked rhodamine-123 efflux capacity. Additionally, P-gp blockade significantly inhibited murine alloimmune T cell activation in a dose-dependent fashion. In vivo, P-gp blockade significantly prolonged graft survival in Balb/c recipients of C57BL/6 cardiac allografts from 8.5 ± 0.5 to 11.7 ± 0.5 days (P < 0.01), similar in magnitude to the effects of monotherapy with cyclosporine A. Moreover, P-gp blockade, compared to controls, attenuated intragraft expression of CD3 and CD80, but not CD86, and inhibited IFN-γ and IL-4 production (P < 0.05). In the setting of systemic CD86 inhibition, P-gp blockade suppressed IFN-γ and IL-4 production significantly further (to 98% and 89% inhibition, respectively) compared to either P-gp or anti-CD86 blockade alone, and markedly prolonged allograft survival compared to anti-CD86 blockade alone (40.5 ± 4.6 versus 22.5 ± 2.6 days, respectively, P < 0.01). Our findings define a novel in vivo regulatory role of P-gp in alloimmunity and identify P-gp as a potential therapeutic target in allotransplantation.     

Synthesis and activity of tetrapeptidic HTLV-I protease inhibitors possessing different P3-cap moieties

The causative agent behind adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy is the human T-cell leukemia virus type 1 (HTLV-I). Tetrapeptidic HTLV-I protease inhibitors were designed on a previously reported potent inhibitor KNI-10516, with modifications at the P(3)-cap moieties. All the inhibitors showed high HIV-1 protease inhibitory activity (over 98% inhibition at 50nM) and most exhibited highly potent inhibition against HTLV-I protease (IC(50) values were less than 100nM).     

Cloning and expression analysis of novel Aux/IAA family genes in Gossypium hirsutum

The associations between polymorphisms of prostate stem cell antigen (PSCA-rs2294008C > T and -rs2976392G > A) and gastric cancer (GC) risk for Eastern Asians have been commonly studied, but the results were conflicting. The aim of the present study was to further assess the associations by the method of meta-analysis. The databases of Medline, Embase and CNKI (up to May 25th, 2011) were retrieved to identify eligible case-control studies. Odds ratio (OR) and 95% confidence interval (95%CI) were used to present the strength of the associations. In total, eight case-control studies in seven articles with 16792 individuals (9738 cases of GC and 7054 controls) were included in this meta-analysis. Through quantitative analyses, we found that T allele of rs2294008C > T and A allele of rs2976392G > A were significantly associated with increased GC risk [rs2294008C > T: OR (95%CI) = 1.31 (1.22-1.42), P_z-test < 0.001, P_{heterogeneity} = 0.166 for TT vs. C carriers; rs2976392G > A: OR (95%CI) = 1.36(1.24-1.50), P_z-test = 0.015, P_{heterogeneity} = 0.111 for AA vs. G carriers]. The results of subgroup analyses (according to histopathology, countries and sources of controls) indicated that T allele of rs2294008C > T and A allele rs2976392G > A were associated with increased risk of both intestinal- and diffuse-type GC, and associated with increased risk of GC for Chinese, Japanese, Koreans, PCC and HCC/PHCC. Furthermore, T allele of rs2294008C > T was also associated with increased risk of cardia and non-cardia GC, and associated with increased risk of GC for males and females. Besides those, this meta-analysis also indicated that the interactions between T allele of rs2294008C > T and A allele of rs2976392G > A was associated with increased risk of GC (A-T vs. G-T: OR = 1.16, 95%CI = 1.06-1.27, P_z-test = 0.001, P_{heterogeneity} = 0.835). Although modest limitations and potential bias cannot be eliminated, this meta-analysis suggests that PSCA -rs2294008C > T and -rs2976392G > A are potential factors of GC development for Eastern Asians, and future work may incorporate these findings and evaluate these variants as potential markers for screening and early diagnosis of GC.     

Identification of HTLV-I gag protease and its sequential processing of the gag gene product

The full-length provirus of human T-cell leukemia virus type I (HTLV-I) was isolated from MT-2, a lymphoid cell line producing HTLV-I. In transfected cells, structural proteins of HTLV-I, the gag and env products, were formed and processed in the same manner as observed in MT-2 cells. The nucleotide sequence was determined for a region between the gag and pol genes of the proviral DNA clone containing an open-reading frame. The deduced amino acid sequences show that this open-reading frame encodes a putative HTLV-I protease. The protease gene (pro) of HTLV-I was investigated using a vaccinia virus expression vector. Processing of 53k gag precursor polyprotein into mature p19, p24, and p15 gag structural proteins was detectable with a recombinant plasmid harboring the entire gag- and protease-coding sequence. We demonstrated that the protease processed the gag precursor polyprotein in a trans-action. A change in the sequence Asp(64)-Thr-Gly, the catalytic core sequence among aspartyl proteases, to Gly-Thr-Gly was shown to abolish correct processing, suggesting that HTLV-I protease may belong to the aspartyl protease group. The 76k gag-pro precursor polyprotein was identified, implying that a cis-acting function of HTLV-I protease may be necessary to trigger the initial cleavage event for its own release from a precursor protein, followed by the release of p53 gag precursor protein. The p53 gag precursor protein is then processed by the trans-action of the released protease to form p19, p24, and p15.     

Assessment of clinical outcomes in breast cancer patients treated with taxanes: multi-analytical approach

Polymorphisms in genes encoding CYPs (Phase I) and ABCB1 (Phase III) enzymes may attribute to variability of efficacy of taxanes. The present study aims to find the influence of CYP and ABCB1 gene polymorphisms on taxanes based clinical outcomes. 132 breast cancer patients treated with taxanes based chemotherapy were genotyped for CYP3A4*1B, CYP3A5*3, CYP1B1*3, CYP2C8*3, ABCB1 1236C>T, 2677G>T/A and 3435C>T polymorphisms using PCR-RFLP. Associations of genetic variants with clinical outcomes in terms of response in 58 patients receiving neo-adjuvant chemotherapy (NACT), and chemo-toxicity in 132 patients were studied. Multifactor dimensionality reduction (MDR) analysis was performed to evaluate higher order gene–gene interactions with clinical outcomes. Pathological response to taxane based NACT was associated with GA genotype as well as A allele of CYP3A5*3 polymorphism (P_corr = 0.0465, P_corr = 0.0465). Similarly, association was found in dominant model of CYP3A5*3 polymorphism with responders (P_corr = 0.0465). Haplotype analysis further revealed A_CYP3A4–A_CYP3A5 haplotype to be significantly associated with responders (P_corr = 0.048). In assessing toxicity, significant association of variant (TT) genotype and T allele of ABCB1 2677G>T/A polymorphism, was found with ‘grade 1 or no leucopenia’ (P_corr = 0.0465, P_corr = 0.048). On evaluating higher order gene–gene interaction models by MDR analysis, CYP3A5*3; ABCB11236C>T and ABCB1 2677G>T/A; ABCB1 3435C>T and CYP1B1*3 showed significant association with treatment response, grade 2–4 anemia and dose delay/reduction due to neutropenia (P = 0.024, P = 0.004, P = 0.026), respectively. Multi-analytical approaches may provide a better assessment of pharmacogenetic based treatment outcomes in breast cancer patients treated with taxanes.     

Characterization of T cell clones specific to a determinant of hepatitis B virus core and e antigens in chronic type B hepatitis: Implication for a T cell mechanism of HBV immunopathogenesis

T cell clones specific for hepatitis B core (HBcAg) and e (HBeAg) antigens of hepatitis B virus (HBV) were generated from liver infiltrates of HBeAg-positive patients. Analyzed with a panel of overlapping synthetic peptides spanning the complete sequences of HBcAg and HBeAg, eight clones responded specifically to the e2 peptide (PAYRPPNAPIL; amino acid residues 130–140 of HBcAg and HBeAg), which was doubly restricted by class I and II molecules. A preferential usage of the T cell receptor (TCR) chain variable (V) gene was found: V12.1 for five HLA-Cw9(3)-restricted cytotoxic T lymphocyte (CTL) clones, and V7.1 for three other HLA-DRw52-restricted type 1 helper T cell (Th1) clones. Although heterogeneous in the usage of TCR chain joining region (J) segments, their junctional-region sequences revealed conserved hydrophilic serine residues in seven of the eight e2-specific T cell clones. Single alanine substitution of the centrally located and the only hydrophilic asparagine residue of e2 peptide abrogated T cell responsiveness. The nonstimulatory e2 analogue could competitively inhibit e2-specific responses. These results demonstrate that both CTL and Th1 clones recognizing a determinant of HBcAg and HBeAg are present in the liver of chronic hepatitis B patients. The preferential V gene usage and the expression of conserved residues in junctional-region sequences of TCR chains by viral-peptide-specific, intrahepatic T cells may provide a T cell mechanism of HBV immunopathogenesis.     

Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen

A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66 kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 °C after 1 h and acts optimally at 70 °C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0 M urea and 3.0 M guanidine hydrochloride) and organic solvents [CH₃CN-H₂O (1:1, v/v) and MeOH-H₂O (1:1, v/v)] when incubated for 1 h. The enzyme showed a high resistance to autodigestion even at low concentrations.     

Profilin binding to sub-micellar concentrations of phosphatidylinositol (4,5) bisphosphate and phosphatidylinositol (3,4,5) trisphosphate

Profilin is a small (12-15 kDa) actin binding protein which promotes filament turnover. Profilin is also involved in the signaling pathway linking receptors in the cell membrane to the microfilament system within the cell. Profilin is thought to play critical roles in this signaling pathway through its interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] (P.J. Lu, W.R. Shieh, S.G. Rhee, H.L. Yin, C.S. Chen, Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity, Biochemistry 35 (1996) 14027-14034). To date, profilin's interaction with polyphosphoinositides (PPI) has only been studied in micelles or small vesicles. Profilin binds with high affinity to small clusters of PI(4,5)P₂ molecules. In this work, we investigated the interactions of profilin with sub-micellar concentrations of PI(4,5)P₂ and PI(3,4,5)P₃. Fluorescence anisotropy was used to determine the relevant dissociation constants for binding of sub-micellar concentrations of fluorescently labeled PPI lipids to profilin and we show that these are significantly different from those determined for profilin interaction with micelles or small vesicles. We also show that profilin binds more tightly to sub-micellar concentrations of PI(3,4,5)P₃ (K_D = 720 μM) than to sub-micellar concentrations of PI(4,5)P₂ (K_D = 985 μM). Despite the low affinity for sub-micellar concentration of PI(4,5)P₂, profilin was shown to bind to giant unilamellar vesicles in presence of 0.5% mole fraction of PI(4,5)P₂ The implications of these findings are discussed.     

Discovery of potent sulfonamide P4-capped ketoamide second generation inhibitors of hepatitis C virus NS3 serine protease with favorable pharmacokinetic profiles in preclinical species

Hepatitis is a disease characterized by inflammation of the liver, usually producing swelling and, in many cases, permanent damage to liver tissues. Viral hepatitis C (HCV), a small (+)-RNA virus, infects chronically 3% of the world’s population. Boceprevir, SCH 503034, (1) our first generation HCV inhibitor, has already established proof-of- concept and is currently in late stage (phase III) clinical trials. In view of the positive data from our first generation compound, further work aimed at optimizing its overall profile was undertaken. Herein, we report that extension of our earlier inhibitor to the P₄ pocket by introducing a new sulfonamide moiety and optimization of the P1/P₁′ capping led to the discovery of a novel series of inhibitors of the HCV NS3 serine protease. Optimization of the P₁ residue significantly improved potency and selectivity. The combination of optimal moieties led to the discovery of compound 47 which, in addition to being a potent inhibitor of HCV subgenomic RNA replication, was also found to have good PK profile in rat, dog and monkey.     

An updated meta-analysis of endothelial nitric oxide synthase gene: Three well-characterized polymorphisms with ischemic stroke

Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene may influence the risk of ischemic stroke (IS), but the results are still debatable. A meta-analysis was performed to investigate the association between the eNOS gene polymorphisms in IS risk. Case–control studies on the association between the G894T, T-786C, and 4b/a polymorphisms and IS were searched up to July 2012, and the genotype frequencies in the control group were found to be consistent with the Hardy–Weinberg equilibrium (HWE). The effect summary odds ratio (OR) and 95% confidence intervals (CIs) were obtained. Meta-regression was used to explore the potential sources of heterogeneity. Funnel plots and Egger's test was used to estimate small study biases, and heterogeneity was assessed by chi-square-based Q-test and I² test. There were total 6537/6475 cases/controls for G894T, 3459/3951 cases/controls for 4b/a, and 2125/2673 cases/controls for T-786C polymorphism. For G894T and 4b/a, a significant association of 894 T allele and 4a allele with increased risk of IS was found in Asians (TT + GT vs. GG: p < 0.00001, OR = 1.60, 95% CI = 1.38–1.79, P_{heterogeneity} = 0.11; aa + ba vs. bb: P < 0.00001, OR = 1.60, 95% CI = 1.30–1.97, P_{heterogeneity} = 0.02), but not in Caucasians (TT + GT vs. GG: P = 0.60, OR = 0.94, 95% CI = 0.75–1.19, P_{heterogeneity} = 0.002; aa + ba vs. bb: P = 0.13, OR = 0.81, 95% CI = 0.62–1.06, P_{heterogeneity} = 0.63). For T-786C polymorphism, there were no significant differences in genotype distribution between IS and control in Asians (CC + TC vs. TT: P = 0.15, OR = 1.14, 95% CI = 0.95–1.37, P_{heterogeneity} = 0.94) and in Caucasians (CC + TC vs. TT: P = 0.72, OR = 0.96, 95% CI = 0.75–1.22, P_{heterogeneity} = 0.53). This analysis provides strong evidence that the eNOS T-786C gene polymorphism is not associated with IS, the G894T and 4b/a polymorphisms might be associated with IS, at least in Asians.     

Conformational effect of a spin crossover iron(II) complex: Bis[N-(2-methylimidazol-4-yl)methylidene-2-aminoethyl]propanediamineiron(II) perchlorate

A iron(II) complex of the linear hexadentate N₆ ligand H₂L^2-3-2, [Fe(H₂L^2-3-2)](ClO₄)₂, was synthesized and the spin crossover properties were investigated, where H₂L^2-3-2 denotes bis[N-(2-methylimidazol-4-yl)methylidene-2-aminoethyl]propanediamine. The complex showed a gradual and reversible one-step spin crossover (SCO) between the high-spin (S = 2) and low-spin (S = 0) states at T_1/2 = 208 K without hysteresis. The crystal structures were determined at 296 and 250 K (HS state), 230, 210, and 200 K (intermediate between the HS and LS states) and 150 and 110 K (LS state). The spin transition from 296 to 150 K accompanies with the conformation change of the chelate rings at the triamine moiety and the formation of the hydrogen bond network in the same space group of orthorhombic Pbcn (no. 60). However, in the LS state at 110 K, the space group changed from orthorhombic Pbcn at 150 K (Pcan when the same axial setting to 110 K was used) to monoclinic P2₁/a (no. 14) at 110 K, although no spin transition and no change of assembly structure between 150 and 110 K were observed. It give us an idea that the space group transformation is mainly related to the conformational thermodynamic stability of the chelate rings at the triamine moiety and is not directly correlated with the spin transition.     

Population-based and family-based studies on the protein tyrosine phosphatase non-receptor 22 gene polymorphism and type 1 diabetes: A meta-analysis

Purpose

Studies investigating the association between PTPN22 gene C1858T polymorphism and type 1 diabetes (T1D) susceptibility among Caucasian population have reported conflicting results. To investigate this inconsistency, we performed a meta-analysis of all available studies dealing with the relationship between the PTPN22 C1858T polymorphism and T1D.

Methods

Databases including PubMed, Web of Science, and EMBASE were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association.

Results

In total, 33 population-based studies with 22, 485 cases and 35, 292 controls, 9 family-based studies involving 7276 families were included. Under the random-effects model, the per-allele overall OR of the C1858T polymorphism for T1D was 1.89 (95% CI: 1.76–2.02, P < 10^− 5) by pooling all available case–control studies. In addition, we found significant evidence for overtransmission of the risk T allele in family-based studies (overall OR _TDT = 1.58, 95% CI: 1.43–1.74; P < 10^− 5). The summary OR from case–control and family-based association studies was 1.81 (95% CI: 1.70–1.93, P < 10^− 5).

Conclusions

In conclusion, this meta-analysis suggests that C1858T polymorphism in PTPN22 is associated with elevated T1D risk among Caucasian population.