Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases involved in tissue remodeling. They have also been implicated in various disease processes including tumour invasion and joint destruction and are therefore attractive targets for inhibitor design. For rational drug design, information of inhibitor binding at the atomic level is essential. Recently, we have published the refined high-resolution crystal structure of the catalytic domain of human neutrophil collagenase (HNC) complexed with the inhibitor Pro-Leu-Gly-NHOH, which is a mimic for the unprimed (P3-P1) residues of a bound peptide substrate. We have now determined two additional HNC complexes formed with the thiol inhibitor HSCH2CH(CH2Ph)CO-L-Ala-Gly-NH2 and another hydroxamate inhibitor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2, which were both refined to R-values of 0.183/0.198 at 0.240/0.225-nm resolution. The inhibitor thiol and hydroxamate groups ligand the catalytic zinc, giving rise to a slightly distorted tetrahedral and trigonal-bipyramidal coordination sphere, respectively. The thiol inhibitor diastereomer with S-configuration at the P1' residue (corresponding to an L-amino acid analog) binds to HNC. Its peptidyl moiety mimics binding of primed (P1'-P3') residues of the substrate. In combination with our first structure a continuous hexapeptide corresponding to a peptide substrate productively bound to HNC was constructed and energy-minimized. Proteolytic cleavage of this Michaelis complex is probably general base-catalyzed as proposed for thermolysin, i.e. a glutamate assists nucleophilic attack of a water molecule. Although there are many structural and mechanistic similarities to thermolysin, substrate binding to MMPs differs due to the interactions beyond S1'-P1'. While thermolysin binds substrates with a kink at P1', substrates are bound in an extended conformation in the collagenases. This property explains the tolerance of thermolysin for D-amino acid residues at the P1' position, in contrast to the collagenases. The third inhibitor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2, unexpectedly binds in a different manner than anticipated from its design and binding mode in thermolysin. Its hydroxamate group obviously interacts with the catalytic zinc in a favourable bidentate manner, but in contrast its isobutyl (iBu) side chain remains outside of the S1' pocket, presumably due to severe constraints imposed by the adjacent planar hydroxamate group. Instead, the C-terminal Ala-Gly-NH2 tail adopts a bent conformation and inserts into this S1' pocket, presumably in a non-optimized manner. Both the isobutyl side chain and the C-terminal peptide tail could be replaced by other, better fitting groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation caused by persistent inflammatory processes in the airways. An increased cholinergic tone mediates different pathophysiological features of COPD, such as bronchoconstriction and mucus hypersecretion, mostly through activation of the human muscarinic M 3 receptor (hM 3 ) subtype. Tiotropium bromide (Spiriva) is a well established muscarinic antagonist in the pharmacological management of COPD with a once-daily posology. The rationale behind the sustained bronchodilation obtained with tiotropium consists in its slow dissociation from hM 3 receptors. In this study, we performed a comprehensive preclinical comparison of tiotropium with other long-acting muscarinic antagonists (LAMAs) currently in clinical development, namely aclidinium bromide and glycopyrrolate. The different muscarinic antagonists were characterized for their 1) affinity toward the different human muscarinic receptor subtypes expressed in Chinese hamster ovary cells and kinetics of receptor dissociation, 2) potency in inhibiting the agonist-induced activation of muscarinic receptors through measurement of second messengers, and 3) efficacy and duration of bronchoprotection, as tested in a model of acetylcholine-induced bronchoconstriction in anesthetized dogs over a period of 24 h. All of the tested LAMAs showed high affinity and potency toward the hM 3 receptor (tiotropium, pA 2 ϭ 10.4; aclidinium, pA 2 ϭ 9.6; and glycopyrrolate, pA 2 ϭ 9.7). However, dissociation half-lives of the LAMAs from the hM 3 receptor differed significantly (tiotropium, t 1 ⁄2 ϭ 27 h; aclidinium, t 1 ⁄2 ϭ 10.7 h; and glycopyrrolate, t 1 ⁄2 ϭ 6.1 h). In line with their kinetic properties at the hM 3 , the tested LAMAs provided different levels of bronchoprotection in the in vivo setting 24 h after administration (tiotropium ϭ 35%, aclidinium ϭ 21%, and glycopyrrolate ϭ 0% at 24 h) when applied at equieffective doses.
Antagonizing the human M3 muscarinic receptor (hM3R) over a long time is a key feature of modern bronchodilating COPD drugs aiming at symptom relief. The long duration of action of the antimuscarinic drug tiotropium and its kinetic subtype selectivity over hM2R are investigated by kinetic mapping of the binding site and the exit channel of hM3R. Hence, dissociation experiments have been performed with a set of molecular matched pairs of tiotropium on a large variety of mutated variants of hM3R. The exceedingly long half-life of tiotropium (of more than 24 h) is attributed to interactions in the binding site; particularly a highly directed interaction of the ligands' hydroxy group with an asparagine (N508(6.52)) prevents rapid dissociation via a snap-lock mechanism. The kinetic selectivity over hM2R, however, is caused by differences in the electrostatics and in the flexibility of the extracellular vestibule. Extensive molecular dynamics simulations (several microseconds) support experimental results.
Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodeling. They have been implicated in various disease processes including metastasis, joint destruction, and neurodegeneration. Human neutrophil collagenase (HNC, MMP-8) represents one of the three "interstitial" collagenases that cleave triple-helical collagens types I, II, and III. Its 163-residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a noncovalent complex with the hydroxamate inhibitor batimastat. The crystal structure, refined to 2.1 A, demonstrates that batimastat binds to the S1-S2' sites and coordinates to the catalytic zinc in a bidentate manner via the hydroxyl and carbonyl oxygens of the hydroxamate group. The batimastat-collagenase complex is described in detail, and the activities of batimastat analogues are discussed in the light of the protein-inhibitor interactions revealed by the crystallography studies.
A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site (P4-P'+/t0) of type I collagen linked to a C-terminal cystine-knot, and N-terminally extended with (Gly-Pro-Hyp)s triplets for stabilization of the triple-helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triplestranded cystine peptide. While the single chains showed no teodeney to self-association into triple helices, the heterotrimer (cxltz2~xl') was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature (Tin) of
Acetylcholine (ACh), synthesized by choline acetyltransferase (ChAT), and muscarinic M 1 , M 2 , and M 3 receptors (MRs) are involved in fibroblast proliferation. We evaluated ChAT, MRs, and extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor (NF) B activation in lung fibroblasts from patients with chronic obstructive pulmonary disease (COPD), control smokers, and controls. Human fetal lung fibroblasts (HFL-1) stimulated with interleukin (IL)-1, tumor necrosis factor (TNF)-␣, and cigarette smoke extracts (CSEs) were evaluated for ChAT and MR expression. We tested the effects of ACh on fibroblast proliferation and its ability to bind fibroblasts from patients with COPD, control smokers, controls, and HFL-1 stimulated with IL-1, TNF-␣, and CSE. ChAT, M 1 , and M 3 expression and ERK1/2 and NFB activation were increased, whereas M 2 was reduced, in COPD and smoker subjects compared with controls. IL-1 increased the ChAT and M 3 , TNF-␣ down-regulated M 2 , and CSE increased ChAT and M 3 expression while downregulating the expression of M 2 in HFL-1 cells. ACh stimulation increased fibroblast proliferation in patients with COPD, control smokers, and controls, with higher effect in control smokers and patients with COPD and increased HFL-1 proliferation only in CSE-treated cells. The binding of ACh was higher in patients with COPD and in control smokers than in controls and in CSE-treated than in IL-1-and TNF-␣-stimulated HFL-1 cells. Tiotropium (Spiriva; [1␣,2,4,5␣,7-7-hydroxydi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatrcyclo[3.3. [(E)-3-(4-methylphenylsulfonyl)-2-propenetrile, C 10 H 9 NO 2 C], down-regulated the ACh-induced fibroblast proliferation, promoting the MRs and ERK1/2 and NFB pathways involvement in this phenomenon. These results suggest that cigarette smoke might alter the expression of ChAT and MRs, promoting airway remodeling in COPD and that anticholinergic drugs, including tiotropium, might prevent these events.An overpresence of fibroblasts has been observed in chronic airway diseases and several factors, released by both parenchymal and inflammatory cells, can contribute to this event promoting fibroblast proliferation (Jeffery, 1998 ABBREVIATIONS: M 1 , muscarinic M 1 receptor; M 2 , muscarinic M 2 receptor; M 3 , muscarinic M 3 receptor; MR, muscarinic M 1 , M 2 , and M 3 receptor; MAPK, mitogen-activated protein kinase; ACh, acetylcholine; ChAT, choline acetyltransferase; COPD, chronic obstructive pulmonary disease; IL, interleukin; TNF, tumor necrosis factor; CS, cigarette smoke; ERK, extracellular signal-regulated kinase; NF, nuclear factor; CSE, cigarette smoke extract; C, asymptomatic nonsmoking subject(s) with normal lung function; FBS, fetal bovine serum; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; ELISA, enzyme-linked immunosorbent assay; pNFB, phosphorylated NFB; tNFB, total NFB; tiotropium, Spiriva, [1␣,2,4,5␣,7-7-hydroxydi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatrcyclo[3.3. O; 4-DAMP, 4-diphenylacetoxy-N...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.