Peptidylglycine α-Amidating Mono-Oxygenase

Gen. Pharmac. Vol. 31, No. 5, pp. 655–659, 1998 Copyright  1998 Elsevier Science Inc. Printed in the USA.

ISSN 0306-3623/98 $–see front matter PII S0306-3623(98)00192-X All rights reserved

REVIEW

Peptidylglycine ␣-Amidating Mono-Oxygenase: Neuropeptide Amidation as a Target for Drug Design Frank N. Bolkenius* and Axel J. Ganzhorn Synthe´labo Biomole´culaire 16, Rue d’Ankara F67080 Strasbourg Ce´dex, France [Tel: ⫹33 (0)3 88 60 87 99; Fax: ⫹33 (0)3 88 45 90 75] ABSTRACT. 1. Peptidylglycine ␣-amidating mono-oxygenase (PAM) is a bifunctional key enzyme in the bioactivation of neuropeptides. Its biosynthesis, distribution, functional role, and pharmacological manipulation are discussed. 2. PAM biosynthesis from a single gene precursor is characterized by alternative splicing and endoproteolytic events, which control intracellular transport, targeting, and enzyme activity. 3. The enzyme is mainly stored in secretory vesicles of many neuronal and endocrine cells with high abundance in the pituitary gland. Its functional role has been studied using enzyme inhibitors. Thus selective, peripheral PAM inhibition reduces substance P along with an anti-inflammatory action. 4. PAM-related pathologies are characterized by an increased relative abundance of ␣-amidated neuropeptides. To attenuate such hormone overproduction, novel, specific, and disease-targeted PAM inhibitors may be developed based on enzyme polymorphism. gen pharmac 31;5:655–659, 1998.  1998 Elsevier Science Inc. KEY WORDS. Peptidylglycine ␣-amidating monooxygenase, mechanism-based inhibition, neuropeptide, hormone, substance P INTRODUCTION A critical feature of many peptide hormones including thyrotropin-releasing hormone, substance P, gastrin, oxytocin, vasopressin, melanotropin, vasoactive intestinal peptide, neuropeptide Y and adrenocorticotropic hormone, which enables them to mediate intercellular communication, is a carboxyl-terminal ␣-amide group (Eipper et al., 1993). The amidation is catalyzed starting from a glycine-extended prohormone, by the sequential action of two enzymes, peptidylglycine ␣-hydroxylating mono-oxygenase (PHM; EC 1.14.17.3) (Bradbury et al., 1982) and peptidylamido-glycolate lyase (PAL; EC 4.3.2.5) (Katopodis et al., 1990). In mammals both enzymes are derived from a single gene, which in some cases gives rise to a single bifunctional protein, whereas in other cases posttranscriptional and -translational processing leads to the formation of two separate entities (Eipper et al., 1993). The name peptidylglycine ␣-amidating mono-oxygenase (PAM) is used to depict the whole reaction sequence.

First PHM, in a reaction dependent of copper, ascorbic acid, and molecular oxygen (Eipper and Mains, 1988), removes the pro-(S) hydrogen of glycine to form a ␣-hydroxyglycine intermediate with retention of configuration (Ramer et al., 1988; Tajima et al., 1990). There is a close resemblance between PHM and dopamine ␤-mono-oxygenase (D␤M; E.C. 1.14.17.1). Not only the catalyzed reactions are similar, with identical cofactor requirements for both enzymes (Stewart and Klinman, 1991), but there is also a 32% amino acid-sequence homology of their catalytic cores (Southan and Kruse, 1989), suggesting a *To whom correspondence should be addressed. Received 7 April 1998; accepted 18 May 1998.

common evolutionary precursor. The catalytic core of PHM contains two nonequivalent coordinatively bound copper atoms (Yonekura et al., 1996; Boswell et al., 1996; Kolhekar et al., 1997; Prigge et al., 1997), one of which is involved in hydroxyl radical-mediated pro-(S) hydrogen abstraction from the ␣-carbon of the substrate. The ␣-hydroxyglycine intermediate is in turn subject to PALmediated breakdown, producing the amidated peptide plus glyoxylate. Rapid spontaneous cleavage of this intermediate is observed above pH 8, a reaction used in vitro to directly measure PHM activity as amide formation (Tajima et al., 1990). However, in the acidic environment of the secretory granules, PAL is needed because the rate of nonenzymatic N-dealkylation is very slow (Eipper et al., 1993). ␣-Amidated peptides with all 20 amino acids at the C terminus are produced (Tamburini et al., 1988), indicating a broad substrate acceptance by both enzymes, whereas contrasting S2 subsite stereospecificites are observed (Ping et al., 1995). Because ␣-amidation is probably the rate-limiting step in peptide bioactivation (Mains et al., 1991), PAM may provide an interesting point of pharmacological intervention. Due to the great number of possible biological peptide substrates as well as their functional diversity, it appears, nevertheless, difficult to clearly relate a specific pathology to changes in PAM-mediated ␣-amidation. The only significant, disease-related change of PAM activity reported so far is an increase occurring in the cerebrospinal fluid of patients with multiple sclerosis (Tsukamoto et al., 1995). This increase was not correlated with total protein levels in the cerebrospinal fluid or with the level of serum PAM activity, especially during the active disease stage. EXPRESSION AND PROCESSING OF THE PAM PROTEIN

Regional distribution Expression of PAM is high in many neurons and endocrine cells, especially in the pituitary (Braas et al., 1989). In addition, the enzyme is also expressed at high levels in ependymal cells and atrial myo-

656 cytes and at lower levels in astrocytes (Schafer et al., 1992). PAM mRNA transcripts and PAM protein are detected in all major brain areas with the exception of the cerebellum. Very high levels of PAM mRNAs are found in the hypothalamic magnocellular neurons, the hippocampal formation, and olfactory cortex. These areas are also rich in PAM protein. To achieve its function in peptide amidation, PAM must be colocalized with its targets; as a consequence, regions known to contain high levels of amidated neuropeptides also express high levels of PAM mRNA (Braas et al., 1989). Hence, the observed heterogeneous PAM mRNA distribution may reflect regional differences in peptidergic activity. Interestingly, all pyramidal neurons of the hippocampus express very high levels of PAM mRNA, although no identified amidated peptide matches this distribution completely (Eipper et al., 1993). This suggests the occurrence of as yet unidentified amidated neuropeptides in these cells.

Posttranscriptional modifications The product of the gene encoding PAM is subject to numerous posttranscriptional and -translational modifications (Eipper et al., 1993). Alternative RNA splicing generates at least seven forms of PAM protein in the rat, varying from membrane-associated to completely soluble or even inactive proteins, with molecular weights ranging from 120,000 for integral membrane PAM-1, to 42,000 and 35,000 for the catalytic PAL and PHM domains, respectively (Yun et al., 1995; Eipper et al., 1993). The integral PAM molecule contains the catalytic domain of PHM as well as that of PAL, which is situated further to the C terminus. It also contains a protease sensitive C-terminal signal domain, an important determinant for intracellular routing of the PAM molecule. The processing of peptide hormone precursors requires storage in secretory granules together with their modifying enzymes. It is, therefore, important that both enzymes involved in the PAM reaction, PHM and PAL, together reach these granules following their biosynthesis. The integral membrane PAM is subject to endoproteolytic processing at several paired, basic amino acid sites, including the one between the PHM and PAL domains (Perkins et al., 1990), and it appears that soluble PAM proteins are more efficiently targeted to storage granules (Milgram et al., 1994, 1996). In addition, proteolytic processing leads to an enhancement of the enzyme kinetic parameters of PHM without altering those of PAL (Husten and Eipper, 1994). Alternative RNA splicing, in part distinct from that observed with the rat gene, is also found with human PAM (Vos et al., 1995). Thus, the different posttranscriptional and -translational events in conjunction with hormonal regulation of PAM expression (el Meskini et al., 1997) appear to be needed for the selective control of ␣-amidated neuropeptide function. FUNCTIONAL SIGNIFICANCE OF PEPTIDE AMIDATION

Does ␣-amidation always result in a gain of functional potency? The PAM-mediated C-terminal ␣-amidation is required for the full biological potency of numerous peptides, a selection of which has recently been listed along with an index of their estimated gain in functional potency (Merkler, 1994). In most cases the increase in potency is very important, while in a few examples there was no obvious benefit; in no case was the potency decreased. Nevertheless, some recent findings support the idea of a biological function of the glycine-extended precursors, where C-terminal amidation may represent a mechanism for shifting the bioactivity of a given peptide hormone from one target to another. In this regard, it is interesting that the gastrin17-Gly as well as the gastrin17-NH2

F. N. Bolkenius and A. J. Ganzhorn both stimulate DNA synthesis (Seva et al., 1994) and cell growth (Todisco et al., 1995) via distinct receptors or different intracellular mechanisms. Therefore, PAM is not essential in the bioactivation of pancreatic gastrin (Kapuscinski et al., 1995). Another example for a biological function of a glycine-extended precursor peptide is the induction of prolactin secretion by thyrotropin-releasing hormone-Gly in patients with anorexia nervosa, which was not observed in different control groups (Mori et al., 1990).

Inbiting ␣-amidation with disulfiram Dietary copper deficiency in rats leads to decreased in vivo PHM activity (Mains et al., 1985) and is expected to limit neuropeptide ␣-amidation. Copper depletion in whole animals, at the site of enzyme action, appears to be more efficiently achieved by the administration of the copper chelator disulfiram (Antabuse). The disulfiram-lowered in vivo PHM activity entailed diminished tissue levels of amidated ␣-melanotropin and cholecystokinin (Mueller et al., 1993) in heart atrium and pituitary but also in the central nervous system. Moreover, amidated ␣-melanocyte stimulating hormone and joining peptide were diminished in the pituitary (Mains et al., 1986). In contrast, the glycine-extended, inactive precursors of ␣-melanocyte stimulating hormone and joining peptide increased from zero to about one third of the total pituitary content of these peptides. Whereas this result confirms their dependence on PAMmediated, ␣-amidative bioactivation, it also demonstrates that the enzyme is not rate-limiting under normal circumstances. Furthermore, the observed changes led to parallel, time-matched decrements in some behavioral parameters, such as grip strength or balancing on the rotorod (Rahman et al., 1997). Chronic disulfiram administration to rats also results in compensatory increases of PAM activities, being measurable under optimized conditions ex vivo in tissue homogenate (Mains et al., 1986; Mueller et al., 1993), without altering the PAL activity. These increases were due to an enhanced Vmax of the enzymatic reaction rather than increased expression and are related to the copper deficiency.

Modifying substance P with disulfiram The ␣-amidated neuropeptide substance P (SP) is involved in transmission and modulation of sensory information, such as pain, regulation of neuroendocrine processes, and integration of motor function (Pernow, 1983). The synthesis, axonal transport, and release of SP increase during both acute and chronic inflammation, such as rheumatoid arthritis (Colpaert et al., 1983; Gilligan et al., 1994). In contrast, in the central nervous system, SP can have neurotrophic as well as memory-promoting effects (Huston et al., 1993). The number of SP-immunoreactive neurons is decreased in people who died with Alzheimer’s disease (Ang and Shul, 1995). Moreover, the proteolytic degradation of SP is significantly enhanced in the temporal cortex of those victims (Waters and Davis, 1997). Because the glycine-extended SP precursor has an especially high affinity for PHM (Tamburini et al., 1988), it should be an important substrate under physiological conditions. The rate-limiting role of PHM in SP bioactivation was demonstrated using mice (Marchand et al., 1990). Chronic disulfiram administration (100 mg/kg/day, for 12–25 days) produced a significant overall decrease of SP in certain areas of the mouse central nervous system, as well as an increase of its inactive, glycine-extended precursor. The peptide alterations were located in neural elements that usually express SP. As a functional consequence of these altered brain levels, a significant disulfiram-induced increase in tailflick latency is observed, demonstrating an important role of PAM-mediated SP amidation in the generation of pain.

Neuropeptide Amidation DEVELOPING SELECTIVE PAM INHIBITORS Whereas dietary or disulfiram-mediated copper deficiency leads to diminished PHM activity in vivo, this treatment greatly suffers from a lack of specificity because other important enzymes, such as D␤M, cytochrome c oxidase, superoxide dismutase or glutathione peroxidase, and aldehyde dehydrogenase in the case of disulfiram, are likewise affected (Lear and Prohaska, 1997). To better understand the specific functional roles that PAM plays in different tissues, under normal as well as pathological conditions, efforts are being pursued to synthesize selective, cell-penetrating, powerful inhibitors of this enzyme.

4-Phenyl-3-butenoic acid-mediated inactivation The fact that glyoxylic acid phenylhydrazone is a synthetic substrate of PHM (Bradbury and Smyth, 1987) generating oxalic acid phenylhydrazide, led to the identification of 4-phenyl-3-butenoic acid (transstyrylacetic acid; PBA) as a mechanism-based, irreversible inhibitor of porcine pituitary PHM, as well as that of rat thyroid carcinoma cells (Bradbury et al., 1990). The inactivation is time-dependent, depends on the presence of the cofactors Cu2⫹ and ascorbic acid and is prevented in the presence of enzyme substrates. PBA, therefore, fulfills the criteria for an enzyme-activated (mechanismbased) inhibitor. A reactive intermediate may thus be formed during the catalytic process, which binds covalently to a yet unidentified, electron-rich amino acid residue in the catalytic center of PHM, thereby inactivating the enzyme. PBA was able to penetrate cultured thyroid carcinoma cells where it transiently inhibited the intracellular PHM in an ascorbate-dependent manner and was able to reduce the measured content of thyrotropin-releasing hormone (Bradbury et al., 1990). Recently it was shown (Ogonowski et al., 1997), that chronically administered PBA reduces the carrageenan-induced hind paw swelling in rats. This effect correlated with inhibition of serum PAM as well as decreased tissue levels of SP, thus confirming the anti-inflammatory potential of a selective, peripherally acting PAM inhibitor.

Mechanism-based, irreversible PHM inhibitors A number of mechanism-based, irreversible inhibitors of D␤M have been described (Padgette et al., 1985). The close mechanistic analogy between PHM and D␤M, as well as additional structure/activity data (Tamburini et al., 1988; Katopodis and May, 1990), served as rationale for the synthesis of other mechanism-based PHM inhibitors. By using ␣-unsaturated thioacetic acid derivatives, such as transstyrylthioacetic acid, as enzyme-activated inhibitors of horse serum PHM, it could be demonstrated that a radical rather than a reactive sulfoxide was formed as the PHM-inactivating transient species (Casara et al., 1996), similar to what has been postulated for PBA. Another approach resulted in the synthesis of the mechanismbased inhibitor d-Phe-l-Phe-d-vinylglycine (Zabriskie et al., 1992; Andrews et al., 1997). Interestingly, in these two latter studies it has been observed that substitution of the pro-R hydrogen at the ␣-position with a more sterically demanding group decreases or even prevents the inactivation of PHM.

Species-and tissue-selective inhibition with PBA The observed alternative mRNA splicing and posttranslational processing suggest the possibility that PAM proteins with altered properties may occur. A recent study designed for the pharmacological validation of PHM inhibition in rat disease models revealed variable sensitivities towards PBA-mediated inactivation of isozymes isolated from different tissues (Bolkenius et al., 1997). Thus, no time-

657 dependent inactivation is observed with the soluble as well as the membrane-associated PHM from the brain (Fig. 1a). Instead, exclusively reversible, competitive inhibition occurs, confirming the substrate-like binding of PBA to the catalytic center (Fig. 1b). In contrast, the membrane-associated PHM from heart atrium and the soluble isozyme from blood serum are irreversibly inactivated under identical reaction conditions. Similar differences are observed using benzylhydrazine (Bolkenius et al., 1997), a mechanism-based, irreversible inhibitor acting in replacement of the cofactor ascorbic acid, and which forms a free benzyl radical as the inactivating moiety (Merkler et al., 1995). Thus, the PHM isozymes, although kinetically similar with respect to substrate processing, probably display slightly different active-site geometries. These can, in turn, give rise to profound changes in the partition ratios of inhibitory versus noninhibitory pathways of the radicals occurring during the oxidation of PBA or benzylhydrazine. The inactivation kinetics of human and horse serum PHM with PBA and transstyrylthioacetic acid were also studied (Bolkenius et al., 1997). Comparison of the relative inactivation efficiencies demonstrated a decrease in the order of horse ⬎⬎rat⬎human. Speciesand tissue-selective inhibition of PHM thus appears feasible and may be exploited for the specific control of single PAM- dependent hormonal functions.

Specific reversible PAM inhibition Dipeptides containing a C-terminal l-homocysteine residue instead of glycine, and an N-acylated hydrophobic amino acid in the S2 position inhibit PAM with IC50 values in the low nanomolar range (Erion et al., 1994). The inhibitory potency of these compounds is explained by the coordinative binding of the thiol group with the enzyme-bound copper atom involved in the abstraction of the pro-(S) hydrogen (Prigge et al., 1997). Unfortunately, the synthesized compounds penetrated cultured dorsal root ganglion cells only marginally, and micromolar concentrations were needed to obtain a measurable PAM inhibition (Erion et al., 1994). The cell penetration could be somewhat enhanced by using esterified prodrugs, allowing measurable decreases of the intracellular SP (Jeng et al., 1997). Pyruvate-extended N-acetyl amino acids have been synthesized (Mounier et al., 1997) as selective, reversible, competitive inhibitors of PAL. This novel class of compounds has been designed as to resemble in its tautomeric form the ␣-hydroxyglycine-derived intermediate expected to occur transiently in the course of the reaction catalyzed by PAL. Such inhibitors should be useful tools in studying possible regulatory biological activities of ␣-hydroxyglycinated neuropeptides. CONCLUSIONS The pituitary gland is the bidirectional mediator of the hypothalamic regulation of peripheral hormone function. A number of amidated neuropeptides participate in this task, so that PAM, having an especially high activity in the pituitary, appears to be the key enzyme in the correct functioning of this regulatory system. Consequently, its biosynthesis, intracellular transport, and storage in secretory vesicles are carefully controlled. Therefore, when inhibiting the PAM activity in vivo, one must be aware of side effects, resembling those observed during nonselective, longterm inhibition with disulfiram (Rahman et al., 1997). Nevertheless, the anti-inflammatory effect of PBA in carrageenaninduced hind paw swelling supports the view of a disease-promoting role of PAM under these circumstances. This beneficial PBA effect is achieved by a decreased SP-amidation because of inhibition of

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FIGURE 1. a) Effects of 50 ␮M PBA on PHM isolated from rat serum or brain. Whereas time-dependent, irreversible inhibition is observed with the serum PHM, no detectable inactivation is observed with the brain enzyme. Values represent means of six control incubations in the absence of PBA (䉱) and four inactivation experiments with the serum enzyme (䊉)⫾SD. The inactivation kinetics is non-linear. The absence of a time-dependent effect with the brain PHM (䉲) is also found in the presence of 100 ␮M PBA (not shown). b) Reversible, competitive inhibition (Dixon plot) of rat brain PHM with PBA. No time-dependent inactivation occurred. Duplicate samples were run with dansyl-Tyr-Val-Gly as substrate, at concentrations varied from 20 (䊊), 10 (䉲), 7 (䉬), 5 (䉮), 4 (䊉) to 3 (䉫) ␮M (from Bolkenius et al., 1997).

PAM in the periphery (Ogonowski et al., 1997). A localized peripheral action of PBA may be favoured by its selective, enzyme-activated inhibition of the isozymes occurring in serum or certain peripheral tissues, with only reversible inhibition of the enzyme species occurring in brain (Bolkenius et al., 1997). This enzyme polymorphism, together with pharmacokinetic data of the potential drugs, may be used to optimize disease-targeted PAM inhibition and enable us to control the pathology resulting from a local neuropeptide disharmony. In addition, the overall effect of inhibition of ␣-amidation may be very different in normal and disease tissues. PHM is probably not rate-limiting for the small amounts of amidated peptide produced under normal conditions. A higher degree of catalytic-site occupancy because of an accumulation of prohormone, therefore, may compensate for partial enzyme inhibition. In contrast, in a disease situation, substrate saturation of the active site may occur and make the amidation step entirely rate-limiting. For this reason, it is believed that PHM inhibition can be specific in a disease situation. It appears, therefore, necessary to analyse the real impact of peptide ␣-amidation on diseases such as rheumatism (Anichini et al., 1997), inflammatory bowel disease (Holzer et al., 1997), cancer (Martinez et al., 1996), multiple sclerosis (Barker and Larner, 1992), or even anxiety (Fehder et al., 1997). Likewise, it will be useful to study the tissue and cellular distribution of the different PAM isozymes, including their comparative structural and kinetic analysis. Such data will help in the design of novel selective inhibitors. References Andrews M. D., O’Callaghan K. A. and Vederas J. C. (1997) Synthesis of tripeptide inhibitors of peptidylglycine ␣-amidating monooxygenase (PAM) containing d- and l-styrylglycine. Tetrahedron 53, 8295–306. Ang L. C. and Shul D. D. (1995) Peptidergic neurons of subcortical white matter in aging and Alzheimer’s brain. Brain Res. 674, 329–335. Anichini M., Cesaretti S., Lepori M., Maddali-Bongi S., Maresca M. and

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