2. Peptide α-Amidation in Two Steps
2A. The PHM Reaction: Progress in studies on the formation of α-amidated peptides depended crucially on three key findings. First, studies conducted from the 1950’s to the early 1980’s made it clear that the proprotein precursors to peptide-amides always had a Gly residue located to the C-terminal side of the residue that was to be α-amidated (reviewed in (Kumar, Mains, et al., 2016)) (Fig.1A andB ). Second, development of the first useable enzymatic assay of peptide amidation by Bradbury, Finnie and Smyth (A.F. Bradbury, Finnie, & Smyth, 1982). Third, the realization that pituitary cells maintained in serum-free culture medium efficiently stored and secreted POMC products, but were unable to produce α-amidated peptides (Eipper, Glembotski, & Mains, 1983). Taken together, these observations lead to the conclusion that the reaction depended on copper ions, reduced ascorbic acid and oxygen, followed by the purification of PHM, PAL and PAM from a variety of sources (Eipper et al., 1991; Murthy, Keutmann, & Eipper, 1987; Murthy, Mains, & Eipper, 1986; P. P. Tamburini, Young, Jones, Palmesino, & Consalvo, 1990) and the cloning and expression of PAM cDNAs from several species (Bertelsen et al., 1990; Eipper et al., 1987; Glauder, Ragg, Rauch, & Engels, 1990; Mizuno et al., 1987; Ouafik et al., 1992; Stoffers, Green, & Eipper, 1989; Stoffers, Ouafik, & Eipper, 1991). Although the lyase reaction catalyzed by PAL can occur at elevated pH in the absence of enzyme, phylogenetic studies support the presence of a bifunctional, integral membrane PAM protein (Fig.1C ) in the last eukaryotic common ancestor (Bäck, Mains, & Eipper, 2021; Kumar, Mains, et al., 2016).
Most of the enzymological and all of the crystallographic studies of PHM and PAL have been carried out using the soluble, protease-resistant catalytic cores of PHM (PHMcc) and PAL (PALcc) (Fig.1C ). Endoproteolytic digestion of lysates prepared from mammalian cells expressing exogenous PAM and of tissues that express high levels of endogenous PAM revealed that both catalytic activities were remarkably resistant to proteolytic degradation (Husten & Eipper, 1994; Husten, Tausk, Keutmann, & Eipper, 1993). For rat PAM, PHMcc consists of residues 42–356 (Eipper, Quon, Mains, Boswell, & Blackburn, 1995; Kolhekar, Keutmann, Mains, Quon, & Eipper, 1997) and PALcc consists of residues 498–820 (Kolhekar et al., 2002; Kolhekar, Quon, Berard, Mains, & Eipper, 1998). The crystal structures of recombinant PHMcc and PALcc have been extensively studied (Chufan, De, Eipper, Mains, & Amzel, 2009; Prigge, Eipper, Mains, & Amzel, 2004; Prigge, Kolhekar, Eipper, Mains, & Amzel, 1997; Siebert et al., 2005). PAM, PHM, and PAL purified from natural sources and soluble, recombinant PHM, PAL and bifunctional PAM have provided additional insight into the reaction mechanism (Fig.1C ) (Bertelsen et al., 1990; Miller et al., 1992). To date, it has not been possible to obtain high resolution structural data for any bifunctional PAM proteins. Studies of the PHM and PAL components of bifunctional PAM are accomplished by employing the appropriate assays: O2 consumption from a glycine-extended peptide is specific for PHM and glyoxylate production from a carbinolamide is specific for PAL. Although few studies have utilized human PAM, PHM or PAL, the highly conserved nature of the PHM and PAL active sites (Bäck et al., 2021; Kumar, Mains, et al., 2016) suggests that the catalytic properties of all of the various PAM, PHM and PAL proteins mentioned above are quite similar. Readers are encouraged to review the cited references if the exact source of the proteins is important to their research goals. Despite many attempts to express fully active PAM in bacterial, yeast, and insect systems, mammalian cell lines remain the system of choice. PHMcc has been expressed in E. coli with an N-terminal fusion to thioredoxin, but with a yield of only ~100 µg/liter of E. coli (Handa, Spradling, Dempsey, & Merkler, 2012). Production of recombinant rat PHMcc, PALcc and rat MTC PAM in Chinese hamster ovary (CHO) cells has yielded >100 mg of pure enzyme (Bauman, Ralle, & Blackburn, 2007; Miller et al., 1992), sufficient enzyme to support spectroscopic studies (Blackburn, Rhames, Ralle, & Jaron, 2000; Eipper et al., 1995; Evans, Blackburn, & Klinman, 2006; Jaron & Blackburn, 1999), structural biology (Chufan et al., 2009; Prigge et al., 1997; Prigge, Kolhekar, Eipper, Mains, & Amzel, 1999), and amidated peptide production at the multi-gram level (Ray et al., 1993).
PHM function requires two bound copper ions, CuH (with three His ligands) and CuM (with two His and one Met ligand) (Maheshwari et al., 2018; Prigge et al., 2004; Prigge et al., 1999; Siebert et al., 2005). Copper is easily lost from PHM, meaning that assaying PHM activity in crude lysates or in serum samples (Gaier et al., 2012) requires the addition of exogenous copper and analysis of purified PHM and PAM often requires replacement of copper lost during purification (Blackburn et al., 2000; Evans et al., 2006; Jaron & Blackburn, 1999; Murthy et al., 1986). The first step in the amidation reaction is the reduction of Cu(II)H and Cu(II)M to Cu(I) by ascorbate (Fig.2A , Step 1). Studies show that the kinetic mechanism is equilibrium ordered, with the glycine-extended substrate binding first to reduced enzyme (Fig. 2A , Step 2), followed by the binding of O2 to Cu(I)M (Fig.2A , Step 3). Formation of the PHM product, the (S )-α-hydroxyglycine derivative (Cowley, Tian, & Solomon, 2016; Ping, Mounier, & May, 1995), proceeds from the PHM-Cu(I)H-Cu(I)M-substrate-O2complex (Fig. 2A , Step 4) and regenerates oxidized PHM-Cu(II)H-Cu(II)M, preparing the enzyme for the next catalytic cycle (Fig.2A , Step 5).
The chemical mechanism for PHM catalysis has been intensively debated (Chen, Bell, Eipper, & Solomon, 2004; Cowley et al., 2016; W.A. Francisco, Merkler, Blackburn, & Klinman, 1998; Jaron & Blackburn, 1999; Klinman, 2008; Kulathila, Merkler, & Merkler, 1999; Ping et al., 1995); it is agreed that a substrate-based radical on the α-carbon of the glycine is an intermediate and that a copper-superoxo species, CuM(II)-O2, is responsible for hydrogen atom abstraction from the substrate glycine. Recent work from Wu et al. (Wu et al., 2019) suggests that an unusual (μ -O)(μ -OH)Cu(II)MCu(II)His the species responsible for hydrogen atom abstraction from substrate. In addition to the uncertainties about the reduced oxygen species responsible for hydrogen atom abstraction, there remain questions about the structure of the bifunctional PAM, binding site(s) for ascorbate, the electron transfer pathways between the two copper atoms and from the copper atoms to the substrate during catalysis, potential domain motion in PHM to reduce the 11Å distance between the Cu(II), and the possibility of carbinolamide channeling between the PHM and PAL active sites in PAM. Note that development of the current cohort of PHM inhibitors (discussed below) has progressed without definitive answers to these questions. Thus, the PHM and PAL inhibitors developed to date are substrate analogs. The rational design of second and third generation PHM inhibitors would benefit from a resolution of the mechanistic uncertainties, fostering the design of tight-binding transition-state analog inhibitors.
2B. The PAL Reaction: Like PHM, PAL is a metalloenzyme; PAL contains a bound Zn(II) and a bound Ca(II) (Bell et al., 1997; Chufan et al., 2009; De, Bell, Blackburn, Mains, & Eipper, 2006). PAL catalyzes the dealkylation of the (S )-α-hydroxyglycine derivative to the corresponding amide and glyoxylate (Fig.2A , Step 5) (Katopodis, Ping, & May, 1990; Perkins, Husten, & Eipper, 1990). To date, evidence about channeling of the peptidyl-α-hydroxyglycine product from PHM to PAL is conflicting (W.A. Francisco et al., 1998; Moore & May, 1999). One major site for PHM and PAL catalysis is the acidic secretory granules with an internal pH~5 (Mains & May, 1988). The α-hydroxyglycine derivative (a carbinolamide) is stable at this pH (Bundgaard & Kahns, 1991); thus, PAL is required for effective amide formation. The bound Ca(II) has a structural role in PAL, while the bound Zn(II) is likely involved in catalysis (Chufan et al., 2009; Takahashi et al., 2009). The precise role played by Zn(II) is not yet clear; suggestions include Zn(II)-bound water or hydroxide serving as a general base for proton abstraction from the hydroxyl group (Bell et al., 1997; Takahashi et al., 2009) and Zn(II)-coordination to the hydroxyl group to lower its pKa, facilitating proton transfer to a tyrosyl phenolate in the active site (Chufan et al., 2009). Model studies by Tenn et al . (Tenn et al., 2007) suggest that carbinolamide dealkylation is not base-catalyzed and is independent of the pKa of the hydroxyl group of the carbinolamide. Instead, these authors argue that carbinolamide dealkylation is dependent upon the nucleofugality of the departing amidate. If so, the PAL-bound Zn(II) could assist in the departure of the amidated product. Additional studies of PAL catalysis are required to address these mechanistic questions.
In the few cases examined, tissue lysates contained substantially more α-amidated peptide than peptidylglycine precursor and levels of peptidyl-α-hydroxyglycine intermediate were well below peptidylglycine levels (Yin et al., 2011). These observations are consistent with the higher Vmax values reported for PAL vs PHM. As discussed below, the PAL domain plays an essential role in the ability of cells to retrieve membrane PAM from the cell surface. This suggests that inhibitors targeted to the active site of PAL might provide an effective means of inhibiting peptide amidation by reducing the amount of PAM available to amidate newly synthesized peptidylglycine substrates entering immature secretory granules. It is intriguing that the PAM loci most frequently associated with human diseases are in the PAL domain, not the PHM domain (Steinhorsdottir et al., 2014; Thomsen et al., 2018).
3.Effective PHM and PAL Inhibitors Were Developed .
For a period of about 25 years, from 1990 to 2015, there was considerable interest in developing PHM inhibitors and inactivators (Ali et al., 2015; Bolkenius, Ganzhorn, Chanal, & Danzin, 1997; F. Cao et al., 2011; A.G. Katopodis & S.W. May, 1990; Langella et al., 2010). The hope was that a PHM-targeted compound would be therapeutically useful in the treatment of human disease and/or would contribute to our understanding of PHM. Compounds that would intercept the radical intermediate were developed (Zabriskie, Cheng, & Vederas, 1992; Zabriskie, Klinge, Szymanski, Cheng, & Vederas, 1994) and PAM labeling with mechanism-based inactivators (suicide substrates) was used to try to identify the active site amino acids critical to catalysis. There was less interest in developing PAL-targeted compounds, the thought being that the non-enzymatic dealkylation of the accumulated carbinolamide intermediate would supply sufficient levels of the amidated product to alleviate PAL inhibition; as discussed above, PAL-targeted compounds that altered the trafficking of PAM could still prove useful in manipulating amidation in vivo .
Many drugs exhibit cell toxicity. Such therapeutics have greater toxicity towards diseased cells relative to healthy cells and must be used properly in treating a disease. In addition, ongoing research will lead to “molecular zip codes”, a protein or a small molecule that binds tightly and specifically to uniquely target diseased cells (Enbäck & Laakkonen, 2007). The attachment of a drug to a molecular zip code would enable the delivery of the drug specifically to the diseased cells – a strategy enabling the safe use of highly toxic drugs to treat human disease. A toxic PHM-specific inhibitor could be valuable therapeutically if appended to the appropriate molecular zip code. The production of amidated autocrine growth factors by tumor cells suggests that high-affinity cell-impermeant PHM inhibitors might prove useful in controlling the growth of these cells. Most neurons and endocrine cells cleave PHM from PAL within secretory granules, and release the PHM along with peptide. These cells would presumably ingest less of the toxic PHM-specific inhibitor than would cancerous cells, since tumor cells usually leave much of their PAM intact while making the amidated autocrine growth factors which enhance tumorigenicity.
Another use of biomedical significance for a PHM-targeted compound would be as a PAM-specific imaging agent. PHM is a biomarker for specific cancers (Jiménez et al., 2003; Rocchi et al., 2004; Scopsi et al., 1998; Thouënnon et al., 2007), post-polio syndrome (Gonzalez et al., 2009), and neural dysfunction (Bousquet-Moore et al., 2010). In sum, the toxicity concern about a PHM inhibitor/inactivator is short-sighted and is limiting work to develop PHM-dependent imaging agents and provides few options when a PHM-specific molecular zip code becomes available.
3A. PHM Inhibitors: The inhibitory effects of divalent metal ion chelators on peptide amidation became apparent as soon as different buffers were tested for use in enzyme assays of tissue lysates (Eipper, Mains, & Glembotski, 1983). Inhibition by diethyldithiocarbamate (DDC) was reversed only by the addition of Cu(II), providing the first evidence that PAM/PHM catalysis was copper-dependent; no other divalent metal, including Zn(II), restored activity (Eipper, Mains, et al., 1983; Eipper, Park, Keutmann, & Mains, 1986). Other metal chelators like EDTA (Merkler, Kulathila, Young, Freeman, & Villafranca, 1993) and disulfiram inhibit PAM/PHM reversibly (Mains, Park, & Eipper, 1986). Disulfiram (Antabuse) is used for the treatment of alcohol abuse (Kranzler & Soyka, 2018) and has anti-cancer activity (Corsello et al., 2020). The possible link between the anti-cancer activity and PHM inhibition has not been directly investigated. Disulfiram has been used to inhibit α-amidated peptide production in cultured mammalian cells and in rats (Mains et al., 1986; Mueller & Altarac, 1995; Rondeel et al., 1995).
Most of the PHM inhibitors developed to date likely bind to the peptide substrate site within the PHM active site (Prigge et al., 1997). A review of the substrates oxidized by PHM is required to provide context for the PHM inhibitors. Only peptides with a C-terminal glycine are PHM substrates in vivo (K.A. Johnson, Paisley-Flango, Tangarone, Porter, & Rouse, 2007). Synthetic peptides with a C-terminal D-Ala are substrates leading to the corresponding α-amidated peptide and pyruvate (Landymore-Lim, Bradbury, & Smyth, 1983). However, the D-Ala-extended peptides are relatively poor substrates with low Vmax/Km ratios. The Vmax/Km ratio provides an estimate of the first-order rate constant for the conversion of substrate to product and is obtained using a substrate concentration far below its Km value; when comparing different substrates, it provides a measure of enzyme specificity (K. A. Johnson, 2019). For example, the Vmax/Km ratio forN -dansyl-Tyr-Val-D-Ala is 0.06 times the ratio for N-dansyl-Tyr-Val-Gly; the Vmax/Km ratio for N -benzoyl-D-Ala is 0.0005 times the ratio forN -benzoylglycine (Merkler et al., 2008). Systematic studies to determine the effect of the penultimate amino acid on the kinetics of amidation showed that glycine-extended peptides with a penultimate aromatic, hydrophobic, or sulfur-containing amino acid had the highest Vmax/Km ratios while those with a penultimate Lys or Arg had the lowest Vmax/Km ratios (Merkler et al., 1993; P. P. Tamburini et al., 1990). α-Amidated peptides with a C-terminal amino acid amide for all 20 of the common amino acids are known (Eipper, Stoffers, & Mains, 1992). Studies aimed at producing amidated peptides in cell lines using endogenous or exogenous PAM indicate that the general rules for effective amidation developed by Tamburini et al. are not directly applicable to the conditions encountered as PAM and its substrates move through the secretory pathway in cells (Chew et al., 2005). A free carboxylate is required on the C-terminal glycine for amidation. The methyl or ethyl esters are not substrates, but are inhibitors of relatively low affinity (P.P. Tamburini et al., 1988). Similarly, the amidated products are low affinity inhibitors (Glembotski, Eipper, & Mains, 1984).
α-Amidated peptide hormones range considerably in length, from 3 amino acids for TRH to 52 amino acids for adrenomedullin. Much larger proteins that terminate with a C-terminal amide and generate α-amidated chemomodulatory peptides have been identified in the green algae,Chlamydomonas reinhardtii (Luxmi, Kumar, Mains, King, & Eipper, 2019). When expressed exogenously, for therapeutic purposes, monoclonal antibodies whose heavy chains terminate with a glycine are often α-amidated (Skulj, Pezdirec, Gaser, Kreft, & Zorec, 2014); elimination of the low levels of PAM expressed in the mammalian cells used to produce these monoclonal antibodies eliminates their α-amidation (Skulj et al., 2014). Dipeptides, X-Gly, are poor PHM substrates, exhibiting low Vmax/Km ratios (Wilcox et al., 1999) and, in general, longer peptide substrates exhibit higher Vmax/Km ratios (Chew et al., 2005). Modeling of N -α-acetyl-3,5-diiodotyrosylglycine into the structure of PHMcc indicates that the carboxylate of the glycyl residue forms a salt-bridge with Arg240 (Prigge, Mains, Eipper, & Amzel, 2000). This arginine is highly conserved across PHM sequences and likely provides a rationale for the importance of the free carboxylate for substrate binding. PHMcc is comprised of two domains of approximately the same size that are connected by a single strand; the single copper atom bound to each domain is poised at the edge facing the solvent-filled cleft separating the domains (Prigge et al., 1997). The interiors of both domains are hydrophobic and the copper atoms are separated by 11Å (Prigge et al., 1997). Upon reduction, there is virtually no change in PHMcc structure (Prigge et al., 2004). The large, hydrophobic interdomain region of PHMcc is necessary for the amidation of relatively large peptide substrates and is consistent with a preference for penultimate hydrophobic amino acids in the substrate. How electron transfer and radical stability are accomplished in the large, solvent accessible interdomain region of PHM are amongst the unanswered questions about PHM catalysis. A PHM structure with a large peptide substrate bound might provide insight about these issues.
Strikingly, PHM and PAL will accept a wide variety of non-peptide substrates possessing a moiety that is equivalent to the C-terminal glycine, including the N -acylglycines, hippurate (N -benzoylglycine) and substituted hippurates (Merkler et al., 2008), and the bile acid glycine conjugates (King et al., 2000). A carbinolamide intermediate is known to form as these substrates are converted into the amide and glyoxylate. Consistent with the preference for a hydrophobic penultimate amino acid for peptide substrates, the Vmax/Km ratio for N -acylglycine amidation increases as acyl-chain length increases. Early studies by Katopodis and May demonstrated that PHM could also catalyze sulfoxidation, amine N -dealkylation, and O -dealkylation (A.G. Katopodis & S.W. May, 1990) (Fig. 2B, C, D ). The PHM catalyzed O -dealkylation of the imino-oxy acetates yields the corresponding oximes and glyoxylate in a PAL-independent manner (N.R. McIntyre, Lowe, Battistini, Leahy, & Merkler, 2016; N. R. McIntyre, Lowe, & Merkler, 2009; Schade, Kotthaus, Hungeling, Kotthaus, & Clement, 2009). Scorpions, which rely on a variety of amidated peptides to attack the ion channels of their prey, have retained separate genes encoding integral membrane PAM, soluble PHM and soluble PAL (Delgado-Prudencio, Possani, Becerril, & Ortiz, 2019), leading the authors to speculate that the PHM secreted in scorpion venom might catalyze some of these additional reactions. A complete understanding of PHM catalysis must account for the alternative reactions catalyzed by this enzyme.
A series of N-blocked Phe-(D,L)-homocysteine peptide analogs were synthesized and evaluated as PAM inhibitors (Erion, Tan, Wong, & Jeng, 1994; Jeng, Fujimoto, Chou, Tan, & Erion, 1997) (Fig. 3A ). Members of this series with IC50 values of 8-15 nM possess an N -hydrocinnamoyl, N -2-naphthoyl, orN -2-indolyl moiety conjugated to the Phe residue. Synthesis of the benzyl ester ofN -2-naphthoyl-L-phenylalanyl-(D,L)-homocysteine as a prodrug increased the IC50 from 10 to 8,000 nM, but 10 µM free carboxylate or benzyl ester showed approximately 50% inhibition of PHM in cultured rat dorsal root ganglion neurons, (Jeng et al., 1997). The potency of this series of compounds mirrors the substrate specificity information from the glycine-extended peptides. The partial PHM inhibition observed in the dorsal root ganglion neurons identifies cell penetration as a challenge for the future clinical use of these compounds. The disappointing results observed using dorsal root ganglion neurons for the benzyl ester suggest that the benzyl ester is not the optimal prodrug, at least, for this series of PHM inhibitors. The tight-binding of the homocysteine peptide analogs likely results from the coordination of sulfur atom to CuM (Fig. 3B ). The binding of thiorphan (KM = 82 µM), tiopronin (KM = 33 \(u\)M), and captopril (Ki~100 µM), all compounds with a free sulfhydryl group, was attributed to coordination of the sulfur atom to one of PHM-bound Cu(II) atoms (N. R. McIntyre, Lowe, Chew, Owen, & Merkler, 2006; Mueller, Driscoll, & Eipper, 1999).
One class of compounds investigated as PHM inhibitors are the glycolates, R-CO-O-CH2-COOH; the R-groups described include an acyl group (short- and long-chain), a benzoyl group, a phenylacetyl-group, an N -acetyl-amino acid, andN -acetyl-peptides (Barratt et al., 2004; F. Cao et al., 2011; A.G. Katopodis & S.W. May, 1990; Morris et al., 2012; Ping et al., 1995) (Fig. 3A ). The glycolates are analogs of the glycine-extended substrates and were clear substrate analogs for consideration as inhibitors. The glycolates are PHM-specific and have no effect on PAL (Moore & May, 1999). We have summarized the data for the glycolate and structurally similar inhibitors in Table 1A and, from these data, we note the following trends. A number of the glycolate inhibitors exhibit Ki or IC50 values <5 µM, meaning that this class of compounds represent a good starting point for the future development of higher affinity PHM inhibitors. While the C-terminal glycolate analogs of two α-amidated peptide precursors (oxytocin and calcitonin) exhibit IC50 values of 2-12 µM, the structurally simplerO -acylglycolates, like O -lauroylglycolate (CH3-(CH2)10-CO-O-CH2-COOH), exhibit sub-µM affinities, against PHM proteins from cultured mammalian cells. Thus, a future tight-binding glycolate inhibitor might be possible without the complications associated with peptide synthesis. Another encouraging observation is the differences in affinity between PHM proteins for some of the glycolate inhibitors. For example,O -lauroylglycolate exhibits an IC50 of 60 nM for human lung cancer PHM and an IC50 of 35 µM for frog PHM, a ratio of IC50 values of ~600. This suggests that tissue-, organism-, or disease-state-specific PHM inhibitors may be possible. The stereochemistry of the penultimate group has a dramatic effect on the affinity of the glycolate inhibitors. The ratio of the Ki values for the D-amino acid glycolate/L-amino acid glycolate is 40-50 for theN -acetyl-D-Phe-O-CH2-COOH/N -acetyl-L-Phe-O-CH2-COOH pair and for theN -acetyl-D-Leu-O-CH2-COOH/N -acetyl-L-Leu-O-CH2-COOH pair. An even stronger preference for an L-amino acid at the penultimate position was identified for the glycine-extended peptide substrates;N -Acetyl-L-Phe-Gly binds to PHM ≥700-fold more tightly thanN -acetyl-D-Phe-Gly (Ping et al., 1995). Future tight-binding glycolate inhibitors could take advantage of the stereochemical preference for the penultimate position.
Cao et al. (F. Cao et al., 2011) synthesized a series of thioglycolates, R-CO-S-CH2-COOH, to determine any effect of the sulfur atom on the affinity of the PHM inhibitor (Fig. 3A andTable 1B ). In general, the thioglycolates inhibit with higher affinity, with a decrease in the IC50 value by a factor of 2-25 relative to the corresponding glycolate. The greatest difference was observed for theN -acetyl-Phe-O-CH2-COOH/N -acetyl-Phe-S-CH2-COOH pair, with a ratio of IC50 values > 80 for frog PHM. Again, these results suggest that sufficient differences exist between PHM proteins to enable the design of an inhibitor targeted against a specific PHM.
PHM inhibition by the glycolates and the thioglycolates coupled with the discovery of N -acylglycines as PHM substrates lead to a structurally related set of PHM inhibitors, theS -(thiocarbonyl)thioglycolates (R–CS–S–CH2–COOH) (Fig. 3A andTable 1C ). S -(Thiolauroyl)thioglycolate, CH3-(CH2)10-CS-S-CH2-COOH, is the tightest binding inhibitor within this set of compounds, with a Ki = 540 nM, a relatively high affinity for a ground-state, substrate analog. A direct comparison between structurally related glycolates, thioglycolates, and theS -(thiocarbonyl)thioglycolates to define whether PHM binds most tightly to either the -CS-S- group, the -CO-S- group, or the -CO-O- group is not possible because the PHM protein used each study was different. For example, the differences in Ki or IC50 values for C6H5-CH2-CS-S-CH2-COOH (8 µM for rat MTC PHM), C6H5-CH2-CO-S-CH2-COOH, (20 µM for frog PHM), and C6H5-CH2-CO-O-CH2-COOH (2 µM for human H889 cell PHM) might result from the different sources of PHM. The S -(thiocarbonyl)thioglycolate study does provide a direct comparison of Ki values between rat MTC PHM and German cockroach PHM for six of these inhibitors; differences were found, with C6H5-CH2-CS-S-CH2-COOH binding to cockroach PHM with ~10-fold higher affinity (Table 1 ). Again, these data point towards the future development of inhibitors targeting PHM in a species-specific manner.
In their focus on the synthesis of PHM inactivators designed to capture the substrate-based radical that likely forms during catalysis, the Vederas group identified new PHM inhibitors. Included in this group are D-Phe-L-Phe-α-cyclopropylglycine (Andrews, O’Callaghan, & Vederas, 1997) and tripeptides with a C-terminal D- or L-styrylglycine (Zabriskie et al., 1992) (Fig. 3A ). PHM is inactivated bytrans -4-phenyl-3-buteonate (trans -styrylacetate) (A. F. Bradbury, Mistry, Roos, & Smyth, 1990). The cyclopropylglycine-containing tripeptide does not inactivate PHM and binds with low affinity, Ki > 5 mM, additional evidence that PHM does not readily accommodate any substitution on the glycyl α-carbon. None of the styrylglycine-containing peptides inactivate PHM, but all inhibit with IC50 values of 100 to 450 µM. This indicates that the styryl moiety must be optimally positioned in the PHM active site for inactivation to occur. Even a small repositioning of the styryl group eliminates its ability to inactivate PHM; neither N -acetyl-D- norN -acetyl-L-styrylglycine inactivates PHM. Little difference in the IC50 values of the L- and D-styrylglycine containing inhibitors was found (Zabriskie et al., 1992). Thus, the importance of the stereochemistry at the penultimate position for PHM affinity found for the glycine-extended substrates and the glycolate inhibitors (Ping et al., 1995) was not observed for the styrylglycine-containing inhibitors. The significance of these differences is currently unknown and could be resolved by in silico modeling or crystallographic analysis of appropriate PHM-ligand complexes. The information gained from such studies could prove useful for the future design of tight-binding PHM inhibitors.
One clear outcome from the work carried out on PHM inhibitors is preference for hydrophobicity in groups attached to the glycine or glycine analog. Consistent with this observation are reports of hydrophobic organic acids that inhibit PAM with relatively low affinity Examples from this group of PHM inhibitors include 1-(carboxymethyl)-3,5-diphenyl-2-methylbenzene (IC50 = 550 µM) (Cutler et al., 1998), urocanic acid (KI = 10 mM) (Merkler et al., 2008), and 4-pentenoic acid (IC50 = 60 mM) (Rhodes & Honsinger, 1993).
One other PHM inhibitor, mimosine (Fig.3A ), differs from the rest. Mimosine is a toxic, heterocyclic, non-protein amino acid produced by plants (Nguyen & Tawata, 2016), which is known to inhibit the mechanistically-related enzyme, dopamine β-monooxygenase (Hashiguchi & Takahashi, 1977). Mimosine is competitive vs. ascorbate, determined by varying the concentrations of mimosine and ascorbate at one-fixed concentration of glycine-extended substrate, yielding a KI of 4.0 µM for mimosine (Miller et al., 1992).In silico modeling reveals that mimosine binds to PHM between the two copper atoms, forming hydrogen bonds to the backbone carbonyls of Pro-268 and Leu-270 and to the side chain amide of Gln-269 (Fig.3A ). Additional in silico modeling suggests that mimosine and ascorbate bind at approximately the same site in PHM (Fig.3B ), consistent with mimosine being competitive vs. ascorbate. The appropriate attachment of mimosine (or a mimosine analog) to an inhibitor that binds to the peptide site in PHM could yield a bifunctional inhibitor of high potency (Fig.3C ).
3B. PAL inhibitors: The metals bound to PAL remain bound during its purification. Like PHM, metal chelators can inhibit PAL by removing the bound Zn(II). Activity is restored to inactive apo-PAL by the addition of Zn(II) and other divalent metal ions (Bell et al., 1997). Unlike PHM, PAL does not exhibit stereochemical preference at the penultimate position; the kinetic constants forN -acetyl-L-Phe-α-hydroxyglycine andN -acetyl-D-Phe-α-hydroxyglycine are very similar (Ping et al., 1995). C-Terminal pyruvate-extended amino acids, R-CO-CH2-CO-COOH, are inhibitors of PAL with Ki values >15 µM.N -Acetyl-L-Phe-pyruvate is the best PAL inhibitor reported, with a Ki of 0.24 µM. The pyruvate-extended amino acids weakly inhibit PHM, being competitive with ascorbate and a ~100-fold lower potency (Mounier et al., 1997). The related compound, C6H5-CH=CH-SO-CH2-COOH (trans isomer), shows no inhibition or inactivation of PHM at 3 mM, but was not evaluated as a PAL inhibitor (Casara, Banzhorn, Philippo, Chanal, & Danzin, 1996). We are not aware of any other work to develop a PAL-specific inhibitor.