Proc. Natl. Acad. Sci. USA Vol. 95, pp. 12028–12033, September 1998 Pharmacology Aminophosphinic inhibitors as transition state analogues of enkephalin-degrading enzymes: A class of central analgesics HUIXIONG CHEN,FLORENCE NOBLE,PASCALE CORIC,MARIE-CLAUDE FOURNIE-ZALUSKI, AND BERNARD P. ROQUES* De´partementde Pharmacochimie Mole´culaireet Structurale, U 266 Institut National de la Sante´et de la Recherche Me´dicale–Unite´de Recherche Associe´e D1500 Centre National de la Recherche Scientifique, Unite´de Formation et de Recherche des Sciences Pharmaceutiques et Biologiques, 4, avenue de l’Observatoire, 75270 Paris Cedex 06, France Communicated by Jean-Pierre Changeux, Institut Pasteur, Paris Cedex 15, France, July 20, 1998 (received for review March 31, 1998) ABSTRACT Inhibition of aminopeptidase N and neutral thiorphan, a selective NEP inhibitor, or bestatin, an APN endopeptidase-24.11, two zinc metallopeptidases involved in inhibitor, did not significantly prevent [3H]Met-enkephalin the inactivation of the opioid peptides enkephalins, produces catabolism whereas their combination resulted in a clear potent physiological analgesic responses, without major side- reduction of the peptide degradation. This result has been effects, in all animal models of pain in which morphine is confirmed in vivo. Thus, because of the complementary role of active. Dual inhibitors of both enzymes could fill the gap NEP and APN in enkephalin inactivation, selective inhibition between opioid analgesics and antalgics. Until now, attempts of only one of these peptidases gives weak antinociceptive to find a compound with high affinity both for neutral responses whereas strong analgesic effects can be obtained endopeptidase and aminopeptidase N have failed. We report after complete inhibition of opioid peptide metabolism with here the design of dual competitive inhibitors of both enzymes combination of selective inhibitors (5). This leads us to pro- with KI values in the nanomolar range. These have been pose the concept of ‘‘dual’’ inhibitors. Accordingly, com- 9 9 obtained by selecting R1,R2, and R3 determinants in amin- pounds able to interact with the S1 and S2 subsites of NEP and ophosphinic-containing inhibitors: NH2OCH(R1)P(O)O APN, such as kelatorphan (5, 6) and RB 38A (7), were shown (OH)CH2OCH(R2)CONHOCH(R3)COOH, for optimal rec- to produce strong analgesic responses caused by a large ognition of the two enkephalin inactivating enzymes, whose increase in the extracellular levels of enkephalins (4). How- active site peculiarities, determined by site-directed mutagen- ever, these compounds (Fig. 1), which contain a hydroxamate esis, have been taken into account. The best inhibitors were as zinc chelating group, were unable to cross the blood–brain 103 more potent than described dual inhibitors in alleviating barrier, and their affinities for APN were '1003 lower than ' 29 acute and inflammatory nociceptive stimuli in mice, thus those for NEP (KI 10 M). These problems were recently providing a basis for the development of a family of analgesics overcome by designing ‘‘mixed inhibitor prodrugs’’ made of devoid of opioid side effects. potent and selective thiol-containing inhibitors of APN and NEP linked by a disulfide bond (8), which was shown to be Acute and chronic pain are incapacitating diseases, and an cleaved in brain (9), where the targeted enzymes are localized improvement in their management is a high priority. Two (2). One of these compounds, RB 101, has been studied classes of pain-alleviating substances currently are used in extensively. After i.p. administration, it has been shown by clinic. The first one is constituted by morphine and surrogates, microdialysis in the nucleus accumbens of freely moving rats which are the most potent and useful compounds to reduce that RB 101 induced a long lasting increase in the extracellular severe pain, including pain associated with terminal issues. level of Met-enkephalin-like material (10). Moreover, RB 101 Antalgics including aspirin, paracetamol, and related sub- has proved to be very potent after systemic administration in stances provide the second group. These compounds inhibit all animal models of pain without producing the side effects of the formation of hyperalgesic substances such as prostaglan- morphine (11). RB 101 is now in preclinical studies. Given dins and are efficient in reducing inflammatory pain. However, these promising results, it was of major interest to develop a there is a need for compounds capable of filling the gap single molecule capable of inhibiting both NEP and APN with between opioids and antalgics, which could be used for the nanomolar affinities as achieved for dual inhibitors of the two treatment of postoperative, osteoarticular, and neuropathic endopeptidases NEP and angiotensin-converting enzyme (12). pain as well as pain in children and in the elderly. Previous attempts with hydroxamate or thiol inhibitors were One of the most promising avenues in the search for such unsuccessful mainly because these compounds did not fit compounds is to improve the potency of the physiological perfectly the active site of each enzyme. system of pain control (1), constituted by the endogenous It recently has been confirmed, by site directed mutagenesis opioid peptides, enkephalins which interact with two specific experiments, that the positively charged amino group in APN binding sites, the m and the d receptors, strategically located at substrates or inhibitors is stabilized within the active site of the various levels of nociceptive pathways (2). This can be realized peptidase by interacting with a glutamate residue (13). Taking by inhibition of the membrane-bound zinc metallopeptidases these results into account, we have selected compounds con- involved in the rapid inactivation of the enkephalins. One of taining a phosphinic moiety as a zinc coordinating ligand and these enzymes is neutral endopeptidase-24.11 (NEP, neprily- a free N-terminal amino group for optimal APN binding. sin, EC 3.4.24.11) and other one is an exopeptidase, amino- Numerous specific or multiple-phosphinic inhibitors of zinc metallopeptidases, particularly matrixins, have been described peptidase N (APN, EC 3.4.11.2). Biological studies performed (14), but none of them contains a free amino group that was on rat brain and spinal cord slices (3, 4) have shown that assumed to be detrimental for endopeptidase recognition. In The publication costs of this article were defrayed in part by page charge Abbreviations: NEP, neutral endopeptidase; APN, aminopeptidase N; payment. This article must therefore be hereby marked ‘‘advertisement’’ in ICV, intracerebroventricularly; DGNPA, Dansyl-Gly-(pNO2)Phe- accordance with 18 U.S.C. §1734 solely to indicate this fact. bAla. © 1998 by The National Academy of Sciences 0027-8424y98y9512028-6$2.00y0 *To whom correspondence should be addressed. e-mail: Roques@ PNAS is available online at www.pnas.org. pharmacie.univ-paris5.fr. 12028 Downloaded by guest on October 1, 2021 Pharmacology: Chen et al. Proc. Natl. Acad. Sci. USA 95 (1998) 12029 FIG. 1. Structure of the three families of dual NEPyAPN inhibi- tors. this study, we show that aminophosphinic-containing com- O O pounds of the general formula NH2 CH(R1)P(O) O O (OH)CH2 CH(R2)CONH CH(R3)COOH, in which the side chains have been selected for optimal recognition of NEP and APN, are the first dual competitive inhibitors with nano- molar affinities for both enzymes. As expected, these com- pounds were more potent than described dual APNyNEP inhibitors in both severe and chronic animal models of pain, thus providing a basis for the development of new analgesics fulfilling the gap between antalgics and opioids. MATERIALS AND METHODS Chemicals. Cbz-Phe-His-Leu (Cbz, carbobenzoxy) was from Bachem. Alanine p nitro-anilide was from Sigma, and Dansyl- b Gly-(pNO2)Phe- Ala (DGNPA) was prepared as described FIG. 3. Dixon plot analysis of the inhibitory activity of compound 9B for NEP and APN. The experiments were performed at 25°C in 50 (15). Details on the synthesis of the various inhibitors, sum- z marized in Fig. 2, will be described elsewhere. mM Tris HCl buffer (pH 7.4). (a) The inhibitory potency of 9B for NEP was measured by using DGNPA as substrate at the concentra- Assay For in Vitro NEP and APN Inhibition. NEP was tions indicated (h,5mm; Œ,10mM; E,25mM; ■,50mM; ‚, 100 mM). purified to homogeneity from rabbit kidney (16). IC50 values (b) The inhibitory potency of 9B for APN was measured by using 5 m were determined with DGNPA (Km 37 M) as substrate Ala-PNA as substrate at the concentrations indicated (h,25mM; Œ, according to the procedure described (15). Aminopeptidase 50 mM; E, 100 mM; ■, 200 mM). Each point of the Dixon plots from hog kidney was purchased from Boehringer Mannheim, represents the mean of triplicate determinations. These experiments 6 6 and inhibitory potencies were determined by using L-alanine- were repeated five times, leading to a mean Ki value ( SEM) of 0.80 5 m 0.05 nM for NEP and 2.5 6 0.3 nM for APN. p-nitroanilide (Ala-pNA, Km 400 M) as substrate (17). Dixon plot analyses (Fig. 3 a and b) of the inhibition of both In Vivo Inhibition of NEP. The inhibition of cerebral NEP, enzymes by compound 9B showed that this compound is a induced by i.v. injection of 100 mgykg compound 9B in mice, competitive inhibitor. The IC values of all the inhibitors 50 was evaluated as described (9). Fifteen minutes after injection, tested were transformed into Ki values by the Cheng–Prussof 5 y 1 y mice were anesthetized with chloral hydrate and were fixed by relationship (Ki IC50 1 S Km) (18). transcardial perfusion of paraformaldehyde, followed by phos- phate buffer. Then, the brain was removed, was homogenized in cold, 50 mM TriszHCl buffer, and was incubated with bestatin and captopril and with or without thiorphan. Then, [3H]-D.Ala2-Leu-enkephalin was added, and the amount of [3H]Tyr-D.Ala-Gly was evaluated.