Structural basis of Ac-SDKP hydrolysis by Angiotensin-I converting enzyme

Angiotensin-I converting enzyme (ACE) is a zinc dipeptidylcarboxypeptidase with two active domains and plays a key role in the regulation of blood pressure and electrolyte homeostasis, making it the principal target in the treatment of cardiovascular disease. More recently, the tetrapetide N-acetyl-Ser–Asp–Lys–Pro (Ac-SDKP) has emerged as a potent antifibrotic agent and negative regulator of haematopoietic stem cell differentiation which is processed exclusively by ACE. Here we provide a detailed biochemical and structural basis for the domain preference of Ac-SDKP. The high resolution crystal structures of N-domain ACE in complex with the dipeptide products of Ac-SDKP cleavage were obtained and offered a template to model the mechanism of substrate recognition of the enzyme. A comprehensive kinetic study of Ac-SDKP and domain co-operation was performed and indicated domain interactions affecting processing of the tetrapeptide substrate. Our results further illustrate the molecular basis for N-domain selectivity and should help design novel ACE inhibitors and Ac-SDKP analogues that could be used in the treatment of fibrosis disorders

Kinetic and structural characterisation of amyloid-β peptides hydrolysis by human angiotensin-1-converting enzyme

Angiotensin‐1‐converting enzyme (ACE), a zinc metallopeptidase, consists of two homologous catalytic domains (N and C) with different substrate specificities. Here we report kinetic parameters of five different forms of human ACE with various amyloid beta (Aβ) substrates together with high resolution crystal structures of the N‐domain in complex with Aβ fragments. For the physiological Aβ(1–16) peptide, a novel ACE cleavage site was found at His14‐Gln15. Furthermore, Aβ(1–16) was preferentially cleaved by the individual N‐domain; however, the presence of an inactive C‐domain in full‐length somatic ACE (sACE) greatly reduced enzyme activity and affected apparent selectivity. Two fluorogenic substrates, Aβ(4–10)Q and Aβ(4–10)Y, underwent endoproteolytic cleavage at the Asp7‐Ser8 bond with all ACE constructs showing greater catalytic efficiency for Aβ(4–10)Y. Surprisingly, in contrast to Aβ(1–16) and Aβ(4–10)Q, sACE showed positive domain cooperativity and the double C‐domain (CC‐sACE) construct no cooperativity towards Aβ(4–10)Y. The structures of the Aβ peptide–ACE complexes revealed a common mode of peptide binding for both domains which principally targets the C‐terminal P2′ position to the S2′ pocket and recognizes the main chain of the P1′ peptide. It is likely that N‐domain selectivity for the amyloid peptide is conferred through the N‐domain specific S2′ residue Thr358. Additionally, the N‐domain can accommodate larger substrates through movement of the N‐terminal helices, as suggested by the disorder of the hinge region in the crystal structures. Our findings are important for the design of domain selective inhibitors as the differences in domain selectivity are more pronounced with the truncated domains compared to the more physiological full‐length forms. DATABASE: The atomic coordinates and structure factors for N‐domain ACE with Aβ peptides 4–10 (5AM8), 10–16 (5AM9), 1–16 (5AMA), 35–42 (5AMB) and (4–10)Y (5AMC) complexes have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org/)

Small-molecule inhibitor of OGG1 suppresses pro-inflammatory gene expression and inflammation

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor–α–induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo

Childhood leukaemia in Europe after Chernobyl: 5 year follow-up.

The European Childhood Leukaemia - Lymphoma Incidence Study (ECLIS) is designed to address concerns about a possible increase in the risk of cancer in Europe following the nuclear accident in Chernobyle in 1986. This paper reports results of surveillance of childhood leukaemia in cancer registry populations from 1980 up to the end of 1991. There was a slight increase in the incidence of childhood leukaemia in Europe during this period, but the overall geographical pattern of change bears no relation to estimated exposure to radiation resulting from the accident. We conclude that at this stage of follow-up any changes in incidence consequent upon the Chernobyl accident remain undetectable against the usual background rates. Our results are consistent with current estimates of the leukaemogenic risk of radiation exposure, which, outside the immediate vicinity of the accident, was small

Fragment-based design for the development of N-domain-selective angiotensin-1-converting enzyme inhibitors

Abstract ACE (angiotensin-1-converting enzyme) is a zinc metallopeptidase that plays a prominent role in blood pressure regulation and electrolyte homeostasis. ACE consists of two homologous domains that despite similarities of sequence and topology display differences in substrate processing and inhibitor binding. The design of inhibitors that selectively inhibit the N-domain (N-selective) could be useful in treating conditions of tissue injury and fibrosis due to build-up of N-domain-specific substrate Ac-SDKP (N-acetyl-Ser-Asp-Lys-Pro). Using a receptor-based SHOP (scaffold hopping) approach with N-selective inhibitor RXP407, a shortlist of scaffolds that consisted of modified RXP407 backbones with novel chemotypes was generated. These scaffolds were selected on the basis of enhanced predicted interaction energies with N-domain residues that differed from their C-domain counterparts. One scaffold was synthesized and inhibitory binding tested using a fluorogenic ACE assay. A molecule incorporating a tetrazole moiety in the P 2 position (compound 33RE) displayed potent inhibition (K i = 11.21 + − 0.74 nM) and was 927-fold more selective for the N-domain than the C-domain. A crystal structure of compound 33RE in complex with the N-domain revealed its mode of binding through aromatic stacking with His 388 and a direct hydrogen bond with the hydroxy group of the N-domain specific Tyr 369 . This work further elucidates the molecular basis for N-domainselective inhibition and assists in the design of novel N-selective ACE inhibitors that could be employed in treatment of fibrosis disorders