Vinyl sulfide cyclized analogues of angiotensin II with high affinity and full agonist activity at the AT(1) receptor

Vinyl sulfide cyclized analogues of the octapeptide angiotensin II that are structurally related to the cyclic disulfide agonist c[Hcy(3,5)]Ang II have been prepared. The synthesis relies on the reaction of the mercapto group of a cysteine residue in position 3 with the formyl group of allysine incorporated in position 5 of angiotensin II. A mixture of the cis and the trans isomers was formed, and these were separated and isolated by RP-HPLC. Thus, the three-atom CH(2)[bond]S[bond]S element of the AT(1) receptor agonist c[Hcy(3,5)]Ang II has been displaced by a bioisosteric three-atom S[bond]CH[double bond]CH element. A comparative conformational analysis of the 13-membered ring systems of c[Hcy(3,5)]Ang II and the 13-membered cyclic vinyl sulfides with cis and trans configuration, respectively, suggested that all three systems adopted very similar low-energy conformations. This similarity was also reflected in the bioactivity. Both of the compounds that contained the ring systems encompassing the cis or trans vinyl sulfide elements between positions 3 and 5 exhibited K(i) values less than 2 nM and exerted full agonism at the AT(1) receptor. In contrast, vinyl sulfide cyclization involving the amino acid residues 5 and 7 rendered inactive compounds. The cyclic vinyl sulfides that have agonist activity were both shown to possess low-energy conformers compatible with the previously proposed 3D model for the bioactive conformation of Ang II.     

Nephrotoxicity of chlorofluoroacetic acid in rats.

Dichloroacetic acid (DCA), chlorofluoroacetic acid (CFA), and difluoroacetic acid (DFA) are inhibitors of pyruvate dehydrogenase kinase. DCA is used for the clinical management of congenital lactic acidosis. Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of DCA and CFA, and DCA is a mechanism-based inactivator of GSTZ1-1. In rodents, DCA causes multiorgan toxicities and is hepatocarcinogenic. The toxic effects of CFA, which is an excellent substrate but a poor inactivator of GSTZ1-1, have not been investigated. The objective of this study was to investigate the nephrotoxicity of CFA. Rats given a single dose of 1.5 mmol/kg CFA became anuric and died within 24 h. Urinalysis and light microscopic analysis showed that rats given 0.6-1.2 mmol/kg CFA developed polyuria, glycosuria, and renal proximal tubular damage. Electron microscopic analysis indicated a role for apoptosis in CFA-induced cell death. The nephrotoxicity of CFA was associated with a dose-dependent increase in inorganic fluoride excretion. Treatment of rats with DCA for 5 days to inactivate GSTZ1-1 failed to prevent metabolism of CFA to fluoride and did not block CFA-induced renal damage. A role for GSTZ1-1-catalyzed release of fluoride from CFA is proposed but a role for other enzymes cannot be excluded. DFA, which is not metabolized to fluoride by GSTZ1-1, was given to rats as a control and was also nephrotoxic: rats given 1.2 mmol DFA/kg/day for 5 days had normal urine volumes but showed proximal and distal tubular damage; fluoride excretion was not elevated. The mechanism of DFA-induced nephrotoxicity is not known but appears to differ from that of CFA.     

Surface‐expressed insulin receptors as well as IGF‐I receptors both contribute to the mitogenic effects of human insulin and its analogues

There is a medical need for new insulin analogues. Yet, molecular alterations to the insulin molecule can theoretically result in analogues with carcinogenic effects. Preclinical carcinogenicity risk assessment for insulin analogues rests to a large extent on mitogenicity assays in cell lines. We therefore optimized mitogenicity assay conditions for a panel of five cell lines. All cell lines expressed insulin receptors (IR), IGF‐I receptors (IGF‐IR) and hybrid receptors, and in all cell lines, insulin as well as the comparator compounds X10 and IGF‐I caused phosphorylation of the IR as well as IGF‐IR. Insulin exhibited mitogenicity EC₅₀ values in the single‐digit nanomolar to picomolar range. We observed correlations across cell types between (i) mitogenic potency of insulin and IGF‐IR/IR ratio, (ii) Akt phosphorylation and mitogenic potency and (iii) Akt phosphorylation and IR phosphorylation. Using siRNA‐mediated knockdown of IR and IGF‐IR, we observed that in HCT 116 cells the IR appeared dominant in driving the mitogenic response to insulin, whereas in MCF7 cells the IGF‐IR appeared dominant in driving the mitogenic response to insulin. Together, our results show that the IR as well as IGF‐IR may contribute to the mitogenic potency of insulin. While insulin was a more potent mitogen than IGF‐I in cells expressing more IR than IGF‐IR, the hyper‐mitogenic insulin analogue X10 was a more potent mitogen than insulin across all cell types, supporting that the hyper‐mitogenic effect of X10 involves the IR as well as the IGF‐IR. These results are relevant for preclinical safety assessment of developmental insulin analogues. Copyright © 2014 John Wiley & Sons, Ltd.     

An evaluation of a high‐pressure 11CO carbonylation apparatus

[¹¹C]Carbon monoxide (¹¹CO) is a versatile building block for the synthesis of Positron Emission Tomography (PET) radioligands. However, the difficulty of trapping ¹¹CO in a small solvent volume has limited its utility. We here report an evaluation of a simple, fully automated high‐pressure synthesizer prototype for the use in ¹¹C‐carbonylation reactions. [¹¹C]Carbon monoxide was easily prepared by online reduction of [¹¹C]carbon dioxide using either Mo(s) or Zn(s) as the reducing agent. The conversion yield of ¹¹CO was >99% when zinc was used as the reducing agent, and the corresponding value for Mo was approximately 71%. When the Zn or Mo column was constantly kept under inert atmosphere, no significant decrease in reducing properties was observed for more than 100 ¹¹CO productions. However, in our hands, Mo reductant was much easier to service. A total of nine functional groups were successfully radiolabeled using the ¹¹CO synthesizer prototype. All measured radiochemical yields exceeded 37%, and the ¹¹CO trapping efficiency was generally above 90%, except for the Suzuki coupling where the trapping efficiency was 80%. This high‐pressure synthesizer using [¹¹C]carbon monoxide as the labeling precursor is easy to operate allowing for ¹¹C‐carbonylation reactions to be performed in a high yield and in a routinely fashion.     

5alpha-reductase 2 polymorphisms as risk factors in prostate cancer

Prostate cancer is a significant cause of death in Western countries and is under the strong influence of androgens. The steroid 5alpha-reductase 2 catalyzes the metabolism of testosterone into the more potent androgen dihydrotestosterone in the prostate gland. The enzyme is a target in pharmacological treatment of benign prostatic hyperplasia using specific inhibitors such as finasteride. Makridakis et al. have characterized the V89L and A49T polymorphisms in recombinant expression systems. The L allelic variant has a lower Vmax/Km ratio than the V variant. In the A49T polymorphism, the T variant has an increased Vmax/Km ratio. We performed a population-based case-control study of the impact of the SRD5A2 V89L and A49T polymorphisms on the risk of prostate cancer. We also studied the relation between the genotypes and age at diagnosis, tumor, node, metastasis stage, differentiation grade, prostate specific antigen and heredity. The study included 175 prostate cancer patients and 159 healthy controls that were matched for age. There was an association with SRD5A2 V89L LL genotype and metastases at the time of diagnosis, OR 5.67 (95% CI 1.44-22.30) when adjusted for age, differentiation grade, T-stage and prostate specific antigen. Heterozygous prostate cancer cases that carried the SRD5A2 A49T AT genotype were significantly younger than cases that carried the AA genotype, (mean age 66 years vs 71, P = 0.038). The SRD5A2 V89L and A49T polymorphisms were, however, not associated with altered prostate cancer risk. Further studies of the V89L polymorphism may lead to better understanding of the etiology of prostate cancer metastases.     

Alkylation and inactivation of human glutathione transferase zeta (hGSTZ1-1) by maleylacetone and fumarylacetone

Glutathione transferase zeta (GSTZ1-1) catalyzes the cis-trans isomerization of maleylacetoacetate or maleylacetone (MA) to fumarylacetoacetate or fumarylacetone (FA), respectively. GSTZ1-1 also catalyzes the glutathione-dependent biotransformation of a range of alpha-haloacids, including dichloroacetic acid. The objective of this study was to investigate the mechanism of inactivation of hGSTZ1-1 by MA and FA and to determine the covalent modification of hGSTZ1-1 by MA and FA in the presence and absence of glutathione. MA and FA (0.01-1 mM) inactivated all hGSTZ1-1 polymorphic variants in a concentration- and time-dependent manner, and this inactivation was blocked by glutathione. The C16A mutant of hGSTZ1c-1c was partially inactivated by MA and FA. Electrospray ionization-tandem mass spectrometry and SALSA (Scoring Algorithm for Spectral Analysis) analyses of tryptic digests of hGSTZ1 polymorphic variants revealed that the active site (SSCSWR) and C-terminal (LLVLEAFQVSHPCR) cysteine residues of hGSTZ1-1 were covalently modified by MA and FA. MA and FA adduction resulted in diagnostic 156-Da shifts in the masses of the modified peptide ions and in their MS-MS fragment ions. Alkylation of the active-site cysteine residues, but not of the C-terminal cysteine, was relatively less intense when hGSTZ1-1 polymorphic variants were incubated with MA or FA in the presence of S-methyl glutathione. These data indicate that MA and FA are substrate and product inactivators of hGSTZ1-1 and covalently modify hGSTZ1-1 at the active-site cysteine residue in the absence of glutathione. The observation that inactivation was blocked by glutathione indicates that binding of glutathione to the active site prevents reaction of MA or FA with the active-site cysteine residue. These data also indicate that MA and FA may covalently modify and inactivate other proteins that have accessible cysteine residues and may, thereby, contribute to dichloroacetic acid-induced or hypertyrosinemia type-I-associated toxicities.     

Immunohistochemical localization and activity of glutathione transferase zeta (GSTZ1-1) in rat tissues.

Glutathione transferase zeta (GSTZ1-1) catalyzes the biotransformation of a range of alpha-haloacids, including dichloroacetic acid (DCA), and the penultimate step in the tyrosine degradation pathway. DCA is a rodent carcinogen and a common drinking water contaminant. DCA also causes multiorgan toxicity in rodents and dogs. The objective of this study was to determine the expression and activities of GSTZ1-1 in rat tissues with maleylacetone and chlorofluoroacetic acid as substrates. GSTZ1-1 protein was detected in most tissues by immunoblot analysis after immunoprecipitation of GSTZ1-1 and by immunohistochemical analysis; intense staining was observed in the liver, testis, and prostate; moderate staining was observed in the brain, heart, pancreatic islets, adrenal medulla, and the epithelial lining of the gastrointestinal tract, airways, and bladder; and sparse staining was observed in the renal juxtaglomerular regions, skeletal muscle, and peripheral nerve tissue. These patterns of expression corresponded to GSTZ1-1 activities in the different tissues with maleylacetone and chlorofluoroacetic acid as substrates. Specific activities ranged from 258 +/- 17 (liver) to 1.1 +/- 0.4 (muscle) nmol/min/mg of protein with maleylacetone as substrate and from 4.6 +/- 0.89 (liver) to 0.09 +/- 0.01 (kidney) nmol/min/mg of protein with chlorofluoroacetic acid as substrate. Rats given DCA had reduced amounts of immunoreactive GSTZ1-1 protein and activities of GSTZ1-1 in most tissues, especially in the liver. These findings indicate that the DCA-induced inactivation of GSTZ1-1 in different tissues may result in multiorgan disorders that may be associated with perturbed tyrosine metabolism.     

Low-abundance kutznerides from Kutzneria sp. 744

Five new (5-9) and four known (1-4) kutznerides were isolated from the actinomycete Kutzneria sp. 744. Compounds 1-9 all consisted of a cyclohexadepsipeptide core with the general structure 2-(1-methylcyclopropyl)-D-glycine-(2S,3aR,8aS)-6,7-dichloro-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid-3-hydroxy-D-glutamic acid-O-methyl-L-serine-L-piperazic acid-(S)-2-hydroxy-3,3-dimethylbutyric acid. Compounds 5, 6, and 8 contained erythro-3-hydroxy-D-glutamic acid, whereas 7 and 9 contained the threo isomer and the hydroxy acid was present as (S)-2-hydroxy-3-methylbutyric acid in 5 and 7. The piperazic acid was C-5-N unsaturated and 4-hydroxylated in 6, 4-chlorinated in 8, and C-5-N unsaturated in 9. Minimal inhibitory concentrations for bacteria were found down to 6 microM (Staphylococcus aureus and Erwinia carotovora) and for fungi down to 70 microM (Fusarium culmorum). The trichlorinated 2 and 8 showed the highest antimicrobial activity, whereas 6, with a hydroxylated piperazic acid unit, did not show any inhibition of the pathogens at 230 microM.     

Disease activity level, remission and response in established rheumatoid arthritis: Performance of various criteria sets in an observational cohort, treated with anti-TNF agents

Background  

Most composite indices of disease activity and response criteria in RA have been validated and compared in clinical trials rather than routine care. We therefore wanted to compare the performance of the DAS28, SDAI and CDAI activity indices, their activity states, their response criteria, and also compare with the ACR response criteria in an observational clinical setting.