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.