Substituted Hippurates and Hippurate Analogs as Substrates and Inhibitors of Peptidylglycine α-Hydroxylating Monooxygenase (PHM)

Abstract

Peptidyl α-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of α-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C₆H₅-CO-NH-CH₂-COOH) inhibit the enzyme with K_i values as low as 0.5 μM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH₂-COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH₂-COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations >100-fold excess of their respective K_i values. Comparison of K_i values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed.

1. Introduction

Dozens of α-amidated peptides are known in mammals,1 insects,² and cnidarians.³ The C-terminal α-amide moiety, required for the activity of most α-amidated peptide hormones,⁴ results from the oxidative cleavage of C-terminal glycine-extended precursor by the PAM/PHM/PAL system.⁵ The first step in the amidation reaction is the ascorbate-, copper-, and O₂-dependent hydroxylation of the glycyl α-carbon⁶ followed by the zinc-, calcium-, and iron-dependent dealkylation of the carbinolamide intermediate to the α-amidated peptide and glyoxylate (Fig. 1).⁷ The first step is catalyzed by peptidyl-α-hydroxylating monooxygenase (PHM) and the second step is catalyzed by peptidylamidoglycolate lyase (PAL).⁸ In mammals, PHM and PAL exist both as individual catalytic units and fused together as a bifunctional protein, peptidylglycine α-amidating monooxygenase (PAM). Mammalian PAM is coded for by a single gene; alternative splicing of the PAM mRNA and proteolytic processing of bifunctional PAM can produce the monofunctional enzymes, PHM and PAL.⁹ In insects and cnidarians, bifunctional PAM is not found; only monofunctional PHM and PAL are known, each coded by a distinct gene.¹⁰ Intriguingly, studies of PAM in the mollusk, Lymnae stagnalis, show the expression of a complex protein with four PHM domains per PAL domain in a pentafunctional enzyme.¹¹ The functions underlying the complexities in the various forms of the PAM/PHM/PAL enzymes are not well understood.

Figure 1. The reactions catalyzed by PHM and PAL.

Bifunctional PAM is compromised of the two monofunctional enzymes. The possible role of Fe(III) in PAL catalysis is unclear.

PHM has potential as a novel therapeutic target because of the role played by specific α-amidated peptides in human disease, examples being luteinizing hormone-releasing hormone (LH-RH) and vasoactive intestinal peptide (VIP) in cancer,¹² substance P in rheumatoid arthritis,¹³ and corticotropin-releasing factor (CRF) in anxiety and depression.¹⁴ Inhibition of PAM would generate the glycine-extended precursors which are generally >1000-fold less potent than the mature α-amidated peptides.⁴ However, a drug targeted against PAM/PHM could prove to be unacceptably toxic by blocking the final maturation of all mammalian α-amidated peptide hormones. In fact, homozygous knock-out of the PAM gene leads to embryonic lethity between e14.5 and e15.5 in mice.¹⁵ The possibility that PHM may also be involved in the biosynthesis of oleamide and other bioactive lipid amides only heightens any concern about toxicity of an anti-PHM drug.¹⁶ While toxicity is likely an issue for an anti-PAM/PHM drug, most anticancer drugs are toxic and must be used carefully to minimize damage to healthy cells. The future development of “molecular zip codes” for the targeted delivery of toxic compounds to the site of disease¹⁷ should ameliorate such concerns and could render an anti-PHM compound a valuable weapon for the treatment of human diseases.

Of course, toxicity of an anti-PHM compound could be an asset in the development of a new class of insecticides. In fact, PHM has been suggested as an attractive target for the development of a novel insecticide,¹⁸ an idea supported by work demonstrating that the elimination of PHM expression is lethal in Drosophila.^10b Alignment of PHM sequences from Drosophila and mammalian PHM sequences shows 41% amino acid identity with 52% overall similarity^10b suggesting that there may be sufficient differences between the insect and mammalian enzymes to develop a PHM-directed insecticide with limited human toxicity.

Structure activity studies of PAM/PHM have been devoted to defining the requirement of a C-terminal glycine or D-alanine for catalysis,^19,20 establishing the amino acid preferences at the penultimate and antepenultimate positions for the peptide substrates,²⁰ defining structural parameters for turnover-dependent inactivation by olefinic compounds,²¹ and developing N-substituted dipeptides with a C-terminal homocysteine as low nanomolar inhibitors.²² Since these earlier studies, we²³ and others²⁴ have shown that relatively simple N-acyl- and N-arylglycines are PHM substrates with (V/K)_app values comparable to the peptide substrates and three dimensional structures of oxidized and reduced PHM have been elucidated to provide a framework for structure-activity data.²⁵ In this report, we further define how simple, substrate-like compounds bind to rat PAM with surprisingly strong affinity. The compound with the highest affinity reported here, S-(thiolauroyl)thioglycolate 55, exhibits an K_i,s of 540 ± 50 nM. We further show differences in K_i,s values between rat PAM and insect PAM and that S-(2-phenylthioacetyl)thioglycolate 60 (K_i,s = 7.9 μM to rat PAM) halts the proliferation of cultured human prostate DU 145 cells in a dose dependent manner. These new data, combined with ongoing work to define the transition-state for glycine hydroxylation²⁶ and to develop PAL-specific inhibitors,²⁷ provide a strong basis for the rational design of anti-PAM/PHM/PAL compounds to treat human disease or to control insect pests.

2. Results

2.1. Substituted hippurates and hippurate analogs as substrates

Katopodis and May²⁴ first demonstrated that hippurate 1, 4-methoxyhippurate 20, and 4-nitrohippurate 23 were oxidized by bifunctional PAM to produce the corresponding amide and glyoxylate in a 1:1 stoichiometry. Subsequent work in our laboratory has verified these results^23a,23b and we have further employed hippurate in mechanistic studies of PHM/PAM catalysis because significant kinetic isotope effects are found when using α-dideuterohippurate as a substrate.²⁸ Further investigation into the structure-activity relationships for hippurate oxidation have not been performed to date, despite the ease of synthesis or the commercial availability of many substituted hippurates and hippurate analogs. The information potential of a hippurate SAR study has likely been overlooked because it was thought that relatively small hippurate-based substrates would have limited contacts within the PHM active site, which lies between two ~150-amino acid domains in an open, solvent accessible cleft.^25a,25b The peptide substrates for PHM in vivo can be large, the largest known being C-terminal glycine-extended sorbin (154 amino acids).²⁹

The data presented in Table 1 indicates that hippurate and a number of hippurate analogs stimulate the PAM-dependent consumption of O₂ in a dose-dependent manner. The dependence of the initial rate of O₂ consumption on the initial concentration of substrate, at one fixed concentration of ascorbate and O₂, is well-described by the standard Michealis-Menten equation yielding the steady-state kinetic values for K_M,app, V_M,app, and (V/K)_app included in Table 1. While we have not assayed for glyoxylate or amide formation in each case, previous work on PHM strongly suggests that the binding of the compounds in Table 1 do not simply stimulate the ascorbate-dependent consumption of O₂, but instead the compounds are oxidized to the amide and glyoxylate.^23,24

Table 1.

Hippurate and Hippurate Analogs as Substrates^a

Name	Structure	KM,app (mM)	VM,app (s−1)	(V/K)app (M−1 s−1)	Rel. (V/K)app
2,6-Difluorohippuric acid (3)	R = 2,6-F2(C6H3)	19 ± 4	4.3 ± 0.6	(2.3 ± 0.2) ×102	0.04
Aceturic acid (2)	R = CH3	9.3 ± 0.5	8.0 ± 0.2	(8.6 ± 0.3) × 102	0.1
2-Iodohippuric acid (4)	R = 2-I(C6H4)	3.4 ± 0.4	3.1 ± 0.1	(9.1 ± 0.7) ×102	0.1
tBOC-Gly (5)	R = (CH3)3CO	3.7 ± 0.2	6.3 ± 0.3	(1.7 ± 0.05) × 103	0.3
2-Methylhippuric acid (6)	R = 2-CH3(C6H4)	4.3 ± 0.3	7.9 ± 0.3	(1.8 ± 0.04) ×103	0.3
4-Aminohippuric acid (7)	R = 4-HN(C6H4)	2.6 ± 0.2	5.6 ± 0.2	(2.2 ± 0.1) ×103	0.4
N-(2-Furoyl)glycine (8)		2.7 ± 0.1	7.5 ± 0.07	(2.8 ± 0.1) × 103	0.5
2-Aminohippuric acid (9)	R = 2-H2N(C6H4)	1.4 ± 0.1	4.2 ± 0.1	(2.9 ± 0.1) × 103	0.5
4-Ethylhippuric acid (10)	R = 4-CH3CH2(C6H4)	1.2 ± 0.2	3.6 ± 0.3	(3.1 ± 0.5) × 103	0.5
4-Methylhippuric acid (11)	R = 4-CH3(C6H4)	1.8 ± 0.2	5.7 ± 0.3	(3.2 ± 0.2) ×103	0.5
4-Hydroxyhippuric acid (12)	R = 4-HO(C6H4)	1.7 ± 0.1	5.8 ± 0.1	(3.5 ± 0.2) ×103	0.6
4-Trifluoromethylhippuric acid (13)	R = 4-CF3(C6H4)	0.77 ± 0.1	2.8 ± 0.1	(3.6 ± 0.5) × 103	0.6
3-Chlorohippuric acid (14)	R = 3-Cl(C6H4)	2.2 ± 0.2	7.8 ± 0.3	(3.6 ± 0.2) × 103	0.6
4-Propylhippuric acid (15)	R = 4-CH3(CH2)2(C6H4)	1.2 ± 0.2	5.1 ± 0.4	(4.2 ± 0.6) × 103	0.7
Nicotinuric acid (16)		1.9 ± 0.1	8.0 ± 0.2	(4.2 ± 0.2) × 103	0.7
3-Methylhippuric acid (17)	R = 3-CH3(C6H4)	1.4 ± 0.1	6.4 ± 0.2	(4.7 ± 0.2) × 103	0.8
4-Bromohippuric acid (18)	R = 4-Br(C6H4)	0.52 ± 0.06	2.5 ± 0.09	(4.9 ± 0.4) × 103	0.8
N-(2-Thienylcarbonyl)glycine (19)		1.6 ± 0.1	9.3 ± 0.2	(6.0 ± 0.3) × 103	1.0
Hippuric acid (1)	R = C6H5	1.3 ± 0.04	8.2 ± 0.08	(6.2 ± 0.1) × 103	1.0
4-Methoxyhippuric acid (20)	R = 4-CH3O(C6H4)	0.82 ± 0.04	5.3 ± 0.08	(6.4 ± 0.2) × 103	1.0
4-Chlorohippuric acid (21)	R = 4-Cl(C6H4)	0.62 ± 0.04	4.1 ± 0.1	(6.6 ± 0.2) × 103	1.1
2-Hydroxyhippuric acid (22)	R = 2-HO(C6H4)	0.86 ± 0.02	4.9 ± 0.05	(7.1 ± 0.1) × 103	1.1
4-Nitrohippuric acid (23)	R = 4-O2N(C6H4)	0.47 ± 0.01	4.2 ± 0.04	(8.9 ± 0.2) × 103	1.4
3-Indolylacetylglycine (24)		0.30 ± 0.02	7.3 ± 0.2	(2.5 ± 0.1) × 103	4.0
L-Pyroglutamyl-Gly (25)		0.31 ± 0.03	9.3 ± 0.4	(3.0 ± 0.2) × 103	4.8

^aSteady-state kinetic constants ± S.E. are from computer fits of the initial rate data to eqn. 1.

We found a ~100-fold variation in (V/K)_app values for the compounds listed in Table 1 that results predominately from a ~60-fold variation in the K_M,app values of 18 mM for 2,6-difluorohippurate 3 to 0.3 mM for 3-indolylacetylglycine 24 and L-pyroglutamyl-Gly 25. The variation in the V_M,app values is smaller for the compounds shown in Table 1, only being 2–3-fold. This pattern of results, the variation of (V/K)_app resulting predominantly from the variation in the K_M,app, was also observed for a set of N-acylglycine PAM substrates.23a Mechanistically, these results suggest there is little variation in the rate of product release for hippurate analogs and instead there is likely an effect of substrate structure on the microscopic rate constants leading to the first irreversible step in PHM catalysis.

2.2. Extended hippurate analogs as substrates

The presence of a penultimate hydrophobic amino acid is preferred by PHM. Using a set of peptides of the form N-dansyl-(Gly)₄-X-Gly in which the amino acid at position “X” was substituted with the 20 amino acids typically found in proteins, Tamburini et al. reported that X = Phe, Tyr, or Ile were ranked first, second, and fourth in terms of the their respective (V/K)_app values for amidation.^20b Erion et al. further found that C-terminal homocysteine dipeptide inhibitors, which were N-blocked with a hydrophobic group yielded the tightest binding compounds to PHM.²² The X-ray structure of PHM shows that several hydrophobic amino acids in the active site have extensive contact with the peptide substrate.^25b Based on all of these results, we hypothesized that moving the benzene ring (or other hydrophobic group) away from the amidoacetate moiety would yield a substrate that bound with higher affinity to PAM.

The data presented in Table 2 validate our hypothesis; virtually all of the extended hippurate analogs exhibit a (V/K)_app value that is higher than that for hippurate. Most informative is a set of related compounds that position the benzene ring away from the amidoacetate moiety with an increasingly longer methylene spacer, -(CH₂)_x-, with X = 0–7. The (V/K)_app for O₂ consumption increases ~30-fold from 6.2 ± 0.1 × 10³ M⁻¹ s⁻¹ for hippurate 1 to 2.0 ± 0.2 × 10⁵ M⁻¹ s⁻¹ for 6-phenylhexanoylglycine 50. This set of compounds indicates that there is an optimum position for the benzyl ring as the (V/K)_app for phenaceturic acid 42 is approximately equal to that for 4-phenylbutyrylglycine 43 and the (V/K)_app for 8-phenyloctanoylglycine 46 is ~2.5-fold lower than the value we measured for 6-phenylhexanoylglycine 50.

Table 2.

Extended Hippurate Analogs as Substrates

Name	Structure	KM,app (mM)	VM,app (s−1)	(V/K)app (M−1 s−1)	Rel. (V/K)app
3-(2-Furyl)acryloylglycine (26)		1.2 ± 0.1	3.4 ± 0.1	(2.8 ± 0.1) × 103	0.45
N-(α-Methylhydrocinnamoyl) glycine (27)	R = (C6H5)CH2CH(CH3)	4.1 ± 0.3	18 ± 0.9	(4.3 ± 0.1) × 103	0.69
Acetyl-Gly-Gly (28)	R = CH3CONHCH2	1.5 ± 0.070	8.9 ± 0.2	(5.9 ± 0.2) × 103	1.0
Hippuric acid (1)	R = C6H5	1.3 ± 0.04	8.2 ± 0.08	(6.2 ± 0.1) × 103	1.0
2-Propylmercaptoacetylglycine(29)	R = (CH3)2CHSCH2	0.22 ± 0.01	1.8 ± 0.02	(8.2 ± 0.3) × 103	1.3
N-[(2-Phenylcyclopropyl)carbonyl] glycine (30)		0.90 ± 0.07	10 ± 0.3	(1.1 ± 0.07) × 104	1.8
Hydrocinnamoylglycinea (31)	R = (C6H5)CH2CH2	0.94 ± 0.1 0.55 ± 0.039	9.9 ± 0.4	(1.1 ± 0.06) × 104	1.8
N-[(Benzylmercapto)carbonyl] glycine (32)	R = (C6H5)CH2S	0.059 ± 0.0070	0.69 ± 0.022	(1.2 ± 0.14) × 104	1.9
CBZ-glycine (33)	R = (C6H5)CH2O	0.59 ± 0.02	8.6 ± 0.1	(1.5 ± 0.03) × 104	2.4
Chloroacetyl-Gly-Gly (34)	R = ClCH2CONHCH2	0.42 ± 0.03	8.9 ± 0.2	(2.1± 0.1) × 104	3.3
Isocaproylglycine (35)	R = (CH3)2CHCH2CH2	0.28 ± 0.03	6.6 ± 0.3	(2.3 ± 0.2) × 104	3.7
tBOC-Gly-Gly (36)	R = (CH3)3COCONHCH2	0.30 ± 0.03	7.5 ± 0.2	(2.5 ± 0.1) × 104	4.0
Phenylmercaptoacetylglycin (37)	R = (C6H5)SCH2	0.19 ± 0.02	4.6 ± 0.2	(2.5 ± 0.1) × 104	4.0
Phenylhydantoic acida (38)	R = (C6H5)NH	0.36 ± 0.08 0.45 ± 0.05	9.7 ± 0.9	(2.7 ± 0.4) × 104	4.4
Hippuryl-Gly-Gly (39)	R = (C6H5)CONHCH2-CONHCH2	0.31 ± 0.06	8.8 ± 1.3	(2.9 ± 0.2) × 104	4.7
Cinnamoylglycine (40)	R = (C6H5)CH=CH	0.23 ± 0.02	8.4 ± 0.3	(3.6 ± 0.3) × 104	5.8
3-Phenylthiopropionylglycinea (41)		0.038 ± 0.02 0.023 ± 0.002	1.6 ± 0.2	(4.2 ± 1.4) × 104	6.8
Phenaceturic acid (42)	R = (C6H5)CH2	0.16 ± 0.005	7.4 ± 0.09	(4.6 ± 0.09) × 104	7.4
4-Phenylbutyrylglycine (43)	R = C6H5(CH2)3	0.16 ± 0.02	11 ± 0.4	(7.0 ± 0.7) × 104	11
4-Nitrobenzoyl-Gly-Gly (44)	R = O2NC6H4CONHCH2	0.11 ± 0.007	8.7 ± 0.2	(7.6 ± 0.4) × 104	12
5-Phenylpentanoylglycine (45)	R = C6H5(CH2)4	0.15 ± 0.01	12 ± 0.4	(7.6 ± 0.4) × 104	12
8-Phenyloctanoylglycinea (46)	R = C6H5(CH2)7	0.10 ± 0.009 0.064 ± 0.006	8.0 ± 0.3	(7.6 ± 0.4) × 104	12
2-Pyridylmercaptoacetylglycine (47)		0.036 ± 0.004	3.0 ± 0.1	(8.3 ± 0.7) × 104	13
Phenylthioacetylglycineb (48)		0.024 ± 0.005	2.4 ± 0.2	(1.0 ± 0.2) × 105	16
Hippuryl-Gly (49)	R = (C6H5)CONHCH2	0.078 ± 0.011	12 ± 0.8	(1.6 ± 0.1) × 105	26
6-Phenylhexanoylglycinea (50)	R = C6H5(CH2)5	0.051 ± 0.007 0.043 ± 0.005	11 ± 0.4	(2.0 ± 0.2) × 105	32

^aInhibition of N-dansyl-Tyr-Val-Gly amidation yields the K_i,s value shown in italics.

^bSubstrate inhibition observed for this compound, K_i = 3.8 ± 1.3 mM

Not surprisingly, replacement of a simple saturated methylene linker between the phenyl group and the amidoacetate moiety has an effect on the (V/K)_app for O₂ consumption. The presence of a more peptide-like linker increases the (V/K)_app, most apparent in comparing hippuryl-Gly 49 to 4-phenylbutyrylglycine 43. Replacement of a –CH₂- moiety with a -NH-, -O-, or -S- has a mixed effect on the (V/K)_app, with both increases and decreases being observed. The (V/K)_app values for CBZ-glycine 33, N-[(benzylmercapto)carbonyl]glycine 32, and phenylmercaptoacetylglycine 37 are higher than that for hydrocinnamoylglycine 31, while the value for 2-propylmercaptoacetylglycine 29 is lower than that for isocaproylglycine 35 and the value for phenylhydantoic acid 38 is also lower than the (V/K)_app for phenaceturic acid 42. Previously, we have reported lower (V/K)_app values for methoxycarbonylglycine (CH₃-O-CO-NH₂-CH₂-COOH) relative to propionylglycine (CH₃-CH₂-CO-NH₂-CH₂-COOH) and 5-butylhydantoic acid (CH₃-(CH₂)₃-NH-CO-NH₂-CH₂-COOH) relative to hexanoylglycine (CH₃-(CH₂)₃-CH₂-CO-NH₂-CH₂-COOH).23a

Finally, replacement of the phenyl group with other hydrophobic groups (a furan, an acetyl group, or a branched aliphatic group) also has a mixed effect on the (V/K)_app. The (V/K)_app for isocaproylglycine 35 is higher than hydrocinnamoylglycine 31 while the (V/K)_app is lower when comparing acetyl-Gly-Gly 28 and tBOC-Gly-Gly 36 to hippuryl-Gly 49, 2-propylmercaptoacetylglycine 29 to phenylthioacetylglycine 48, and 3-(2-furyl)acryloylglycine 26 to cinnamoylglycine 40.

The ratio of the (V/K)_app values for the best substrate included in Table 2, phenylhexanoylglycine 50, to the substrate with the lowest (V/K)_app value, 3-(2-furyl)acryloylglycine 26, is about 70. As was observed in Table 1 and with the N-acylglycines, the variation in the (V/K)_app is largely due to differences in the K_M,app values, ranging from 24 μM for phenylthioacetylglycine 48 to 4.1 mM for N-(α-methylhydrocinnamoyl)glycine 27 (a ratio of 170). The V_M,app values only vary about ~20-fold from 0.69 s⁻¹ for N-[(benzylmercapto)carbonyl]glycine 32 to 14 s⁻¹ for N-(α-methylhydrocinnamoyl)glycine 27.

2.3. N-substituted D-alanines as substrates

It has long been known that C-terminal D-alanine extended peptides were also PHM substrates,¹⁹ but this aspect of PHM chemistry has not been explored in detail. The fact that simple N-blocked glycines are substrates (Tables 1 and 2) provided us with an opportunity to examine the structure-function relationships for PHM-mediated oxidation of the N-substituted D-alanines. As shown in Table 3, the simple N-substituted D-alanines, acetyl-D-alanine 51, benzoyl-D-alanine 52, and CBZ-D-alanine 53 are very poor substrates with (V/K)_app values that are 0.05–0.3% of the corresponding N-substituted glycines. In contrast to the simple N-substituted D-alanines, the one C-terminal D-alanine extended peptide included in our study, N-dansyl-Tyr-Val-D-Ala, is a respectable substrate with a (V/K)_app value that is ~6% of the value for N-dansyl-Tyr-Val-Gly. As we have observed before, the relatively low (V/K)_app values for the D-alanine-extended peptides is largely due to a K_M,app effect. The replacement of the pro-R proton of the glycine with a CH₃ group seems to greatly decrease the affinity of PHM for its substrate.

Table 3.

N-Substituted D-Alanines as Substrates^a

Name	Structure	KM,app (mM)	VM,app (s−1)	(V/K)app (M−1 s−1)	$\frac{{(\text{V}/\text{K})}_{\text{app},\text{Gly}}}{{(\text{V}/\text{K})}_{\text{app},\text{D}-\text{Ala}}}$
Acetyl-D-alanine (51)	R = CH3	1400 ± 160	1.3 ± 0.1	0.95 ± 0.05	900
Benzoyl-D-alanine (52)	R = C6H5	84 ± 23	0.27 ± 0.03	3.2 ± 0.6	1,900
CBZ-D-alanine (53)	R = C6H5CH2O	35 ± 5.0	1.4 ± 0.1	41 ± 5	370
N-dansyl-Tyr-Val-D-Alab,c (54)	R = Dansyl-Tyr-Val	0.031 ± 0.004	0.91 ± 0.06	(3.0 ± 0.2) × 104	17

^atBOC-D-alanine is a substrate with a low V/K. At 800 mM tBOC-D-alanine, we obtained a rate of O₂ consumption of 0.63 s⁻¹, which is approximately equal to the rate of O₂ consumption obtained at 15 mM CBZ-D-alanine.

^bKinetics of N-dansyl-Tyr-Val-(D)-Ala amidation were compared directly to N-dansyl-Tyr-Val-Gly amidation, using the exact same reaction conditions and source of PAM. The steady-state kinetic parameters for N-dansyl-Tyr-Val-Gly for this experiment were: K_M,app = 2.7 ± 0.4 μM, V_M,app = 1.4 ± 0.6 s⁻¹, and (V/K)_app = (5.0 ± 0.7) × 10⁵ M⁻¹ s⁻¹.

^cSubstrate inhibition was observed for this compound, K_i = 470 ± 90 μM.

2.4. Inhibition of PHM by analogs of the hippurate-based substrates

Previous work on PHM^22,23d,30 and another copper-dependent, mechanistically-related enzyme, dopamine β-monooxygenase (DβM),31 has indicated that incorporation of a sulfur atom into a hippurate analog is likely to increase the extent of inhibition for any compound developed as a PHM inhibitor. The sulfur atom is thought to either directly interact with enzyme-bound copper leading to the formation of an E·Cu·inhibitor complex or to chelate the enzyme-bound copper to generate inactive copper-free PHM and the Cu·inhibitor complex free in solution.

The three inhibitors reported here which bind with the greatest affinity, 0.5–3.5 μM, all share the replacement of the amidoacetate moiety, -CO-NH-CH₂-COOH, with the (thiocarbonyl)thioglycolate moiety, -CS-S-CH₂-COOH (Table 4). In fact, in all of the inhibitors included in Table 4 with a K_i,s of ≤ 10 μM, have the carbonyl of the amidoacetate moiety replaced with a thiocarbonyl, C=S. The analog of the benzoyl-D-alanine 52 (K_M,app = 84 mM) that incorporated the -CS-S- in place of -CO-NH-, S-(thiobenzoyl)-(R,S)-thiolactate 66, is a surprisingly effective inhibitor with a K_i,s value of 58 μM (Table 4).

Table 4.

PAM Inhibitors

Name	Structure	Ki,s (μM)
S-(Thiolauroyl)thioglycolic acid (55)		0.54 ± 0.05
S-(Phenylthiocarbamoyl)-3-mercaptopropionic acid (56)		2.6 ± 0.7
S-(4-Methylthiobenzoyl)thioglycolic acid (57)		3.5 ± 0.4
S-(Thiobenzoyl)-4-mercapto-4-cyanopentanoic acid (58)		5.7 ± 0.4
N-(Thiobenzoyl)-(D,L)-alanine (59)		7.2 ± 0.9
S-(2-Phenylthioacetyl)thioglycolic acida (60) (2.5% DMSO)		7.9 ± 2.8 13 ± 1
S-(Phenylthiocarbamoyl)thioglycolic acid (61)		8.6 ± 1.4
S-(3-Phenylthiopropionyl)thioglycolic acida (62) (2.5% DMSO)		9.4 ± 0.8 30 ± 11
6-O-Palmitoyl-L-ascorbateb (63)		35 ± 4
S-(Thiobenzoyl)thioglycolic acida (64) (2.5% DMSO)		39 ± 5 49 ± 8
O-(Phenylcarbamoyl)glycolic acid (65)		54 ± 4
S-(Thiobenzoyl)-(R,S)-thiolactic acid (66)		58 ± 9
Glutathione Ethyl Ester (67)		66 ± 8
N,S-Dibenzoyl-L-cysteine (68)		69 ± 7
N-Phenylthiohydantoic acida (69) (2.5% DMSO)		110 ± 20
S-(4-Methylthiobenzoyl)thioglycolic acid ethyl ester (70)		110 ± 10
Salicylhydroxamic acid (71)		300 ± 20
S-Phenylmercaptoacetic Acid (72)		380 ± 40
O-Benzamidoglycolic acid (73)		1,000 ± 60
Pro-Leu-Gly hydroxamic acid (74)		1,000 ± 110
(S)-N-CBZ-4-amino-2-hydroxybutyric acid (75) (CBZ-HABA)		1,400 ± 110
N-(Benzoyl)-D-serine (76)		1,500 ± 150
3-Benzoylpropionic acid (77)		2,900 ± 480
(CBZ-hydrazido)glycine (78)		7,900 ± 980
N-Benzylglycine (79)		18,000 ± 5,100
(Nicotinamidomethyl)phosphonic acidc (80)		>75,000

^aK_M,app for aceturic acid in 2.5% DMSO = 15.0 mM and the K_M,app for N-dansyl-Tyr-Val-Gly in 2.5% DMSO = 14.9 μM. In the absence of DMSO, the K_M,app for aceturic acid = 9.3 mM and the K_M,app for N-dansyl-Tyr-Val-Gly = 3.5 μM. Values obtained in DMSO are shown in italics. In some cases, we obtained K_i,s values in the presence and absence of DMSO. For such compounds, two values are shown with the value in black representing that measured in the absence of DMSO.

^bInhibition constant included in the table is the IC₅₀ because it is not clear that 6-O-palmitoyl-L-ascorbate should be competitive with the aceturic acid of N-dansyl-Tyr-Val-Gly. Preliminary experiments indicate that 6-O-palmitoyl-L-ascorbate is a non-competitive inhibitor vs. N-dansyl-Tyr-Val-Gly and ascorbate at ambient O₂ (Malka and Merkler, unpublished).

^cNo O₂ consumption observed at 0.9 or 28 mM (nicotinamidomethyl)phosphonate. No inhibition of O₂ consumption of 200 μM N-octanoylglycine (K_M,app = 200 μM) was observed at 28 mM (nicotinamidomethyl)phosphonate. A K_i,s of 75 mM would have decreased the initial rate ~15%, which was not seen.

The relative contribution made by the different structural components of the inhibitors and substrates can be estimated from the inhibitors listed in Table 4. For example, the PHM X-ray structure shows that the glycine carboxylate of the substrate interacts with the positively charged guanidinium of Arg-240.^25b Previous data had shown that esterification of the substrate carboxylate decreased the affinity of PHM for the compound approximately 15-fold, as the K_i,s for the methyl ester of N-dansyl-Tyr-Val-Gly was 54 μM compared to a K_M,app of 3.5 μM for N-dansyl-Tyr-Val-Gly.^20a The data included in Table 4 indicate that the contribution to binding made by the substrate carboxylate is not straightforward. Glutathione ethyl ester 67, K_i,s of 66μM, is lower than the K_M,app for glutathione, 130 μM,³⁰ and the K_i,s for salicylhydroxamate 71, 300 μM, is also lower than the K_M,app for salicylurate of 860 μM.^23b Clearly, there must be interactions other than the substrate carboxylate and Arg-240 that are important for binding in the PHM active site. Contributions made by other structural components of the substrates and inhibitors are included in the discussion.

2.5. Inhibition of PAM-catalyzed carbinolamide dealkylation

The recombinant enzyme used in this research is bifunctional rat PAM containing both the monofunctional PHM and PAL domains.^9a This affords us the opportunity to determine whether the inhibitors shown in Tables 1–4 were targeted specifically against PHM or PAL, or inhibited both activities. PHM-specific inhibition is determined by following the ascorbate-dependent consumption of O₂, PAL-specific inhibition is determined by following the ascorbate-independent dealkylation of α-hydroxyhippurate, while the inhibition of N-dansyl-Tyr-Val-Gly amidation represents the inhibition of both PHM and PAL activities (see Fig. 1).

All of the compounds included in Tables 1–4 were screened for inhibition of PAL activity by measuring the amount of glyoxylate produced in a single time interval from 2.0 mM α-hydroxyhippurate at 0 or 750 μM inhibitor. Under these conditions, <0.4 mM glyoxylate was produced in the absence of inhibitor and <5% inhibition of PAL activity was found for all the compounds in Tables 1–4. Thus, the inhibition observed for the compounds included in this study is specific for PHM. There is one exception, S-(phenylthiocarbamoyl)thioglycolate 61. This compound weakly inhibits PAL activity with an IC₅₀ of 3.5 ± 0.2 mM, which is 400-fold higher than the K_i,app for the inhibition of rat PHM. So even for 61, there is a strong preference for PHM.

2.6. In silico modeling of the PHM • substrate and the PHM • inhibitor complexes

Using Glide and Q-site, we docked 79 of the 80 compounds included in Tables 1–4 into the active site of reduced rat PHM. One compound, (nicotinamidomethyl)phosphonic acid 80, would not dock into the active site. This compound showed no inhibition of PHM at 28 mM. For the remaining 79 compounds that would dock into PHM, the Glide emodel values varied 4-fold, from −30 to −120, and the Glide gscore values varied 2.4-fold, from −3.1 to −7.4 (Table S1, supplementary material). The docking values for 72 of the 79 of the compounds were within a more narrow range of Glide emodel values between −55 to −80. Of the seven compounds with Glide emodel values not in the −55 to −80 range, 5 did not terminate in a glycine: N-dansyl-Tyr-Val-D-Ala 54, N-(thiobenzoyl)-(D,L)-alanine 59, glutathione ethyl ester 67, S-(4-methylthiobenzoyl)thioglycolic acid ethyl ester 70, and salicylhydroamic acid 71.

2.7. Inhibition of insect PHM

There has always been a concern that anti-PHM inhibitors would be too toxic for clinical use because such inhibitors would block the biosynthesis of all the α-amidated peptide hormones and the primary fatty acid amides. However, the toxicity of anti-PHM inhibitors could be a tremendous benefit in another venue - the development of novel insecticides because a large majority of insect peptide hormones are α-amidated.2 For this approach to work, there needs to be sufficient differences between mammalian PHM and insect PHM to enable the development of insect-specific anti-PHM inhibitors.

We determined the K_i,s values for seven of the PHM inhibitors from Table 4 for the B. germanica enzyme and have compared these values to those determined for the rat enzyme (Table 5). There are differences in the K_i,s values between B. germanica and rat; the largest difference being a 4–7-fold difference for S-(4-methylthiobenzoyl)thioglycolic acid 57 and for S-(3-phenylthiopropionyl)thioglycolic acid 62. There are also differences between the K_i,s values for the other five compounds included in Table 5, but the magnitude of the differences is smaller.

Table 5.

Comparison of K_i,s Values Between Insect and Mammalian PHM

Compound	Ki,s (μM) vs. B. germanica PHM	$\frac{\text{Rat}{\text{K}}_{\text{i},\text{s}}}{\text{Insect}{\text{K}}_{\text{i},\text{s}}}$
S-(3-Phenylthiopropionyl)thioglycolic acid (62)	0.87 ± 0.11	11
S-(2-Phenylthioacetyl)thioglycolic acid (60)	3.6 ± 0.6	2.1
S-(Thiobenzoyl)thioglycolic acid (64)	92 ± 10	1.9
S-(Phenylthiocarbamoyl)thioglycolic acid (61)	5.4 ± 0.3	1.6
S-(Phenylthiocarbamoyl)-3-mercaptopropionic acid (56)	5.5 ± 0.8	0.47
S-(4-Methylthiobenzoyl)thioglycolic acid (57)	14 ± 1	0.25
S-Phenylmercaptoacetic Acid (72)	56 ± 6	6.9

2.8. Antiproliferative effects of selected PHM inhibitors on human prostate cancer cells (DU 145)

The probability that an American male will die of prostate cancer is ~10%.³² There are two basic types of prostate cancer: androgen-dependent and androgen-independent. Androgen-dependent prostate cancer cells die when deprived of androgen and, consequently, this form of prostate cancer can be successfully treated with luteinizing hormone-releasing hormone (LH-RH) agonists that bind to the androgen receptors.³³ Unfortunately, most patients relapse into androgen-independent prostate cancer with few good options for treatment.³⁴

LH-RH is not the only α-amidated peptide that stimulates the proliferation of prostate cancer. Analogs of both gastrin-releasing peptide (GRP) and growth hormone-releasing hormone (GH-RH) also show promise for the treatment of prostate cancer.³⁵ Instead of an approach that targets individual α-amidated peptide hormones, another strategy to treat prostate cancer that would block the production of all α-amidated peptide hormones would be to inhibit PHM. To this end, we have evaluated the effectiveness of two of the PHM inhibitors of Table 4, S-(2-phenylthioacetyl)thioglycolate 60 (K_i,s = 7.9 μM) and S-(4-methylthiobenzoyl)thioglycolic acid 57 (K_i,s = 3.5 μM), as growth inhibitors for human androgen-independent prostate cancer cells (DU 145 cells).

S-(2-Phenylthioacetyl)thioglycolate 60 shows significant anti-proliferative effects on the growth of DU 145 cells at concentrations >500 μM (Fig. 2). The dose required to inhibit the proliferation of the DU 145 cells is ~60-fold higher than the K_i,s for the inhibition of mammalian PHM. There are many reasons that 60 would be considerably less effective as an anti-proliferative than for the inhibition of PHM in vitro; one possibility being that the inhibitor does not easily cross the cell membrane. To address this possibility, we examined the anti-proliferative activity of a more hydrophobic inhibitor, S-(4-methylthiobenzoyl)thioglycolate 57 and its ethyl ester 70. Despite exhibiting a lower K_i,s in vitro, 57 is less effective than 60 in inhibiting the growth of DU 145 cells, requiring ~1.0 mM to reduce the number of viable cells to 50% (Fig. 3). The ethyl ester 70 shows only ≤10% growth inhibition at 100 μM (Fig. 3).

Figure 2. The effect of S-(2-phenylthioacetyl)thioglycolate 60 on the growth of human prostate DU 145 cells.

Cells were grown to 50,000 cells per well and then treated with the indicated concentration of S-(2-phenylthioacetyl)thioglycolate or N-(4-hydroxyphenyl) retinamide (4-HPR). Cell growth was measured at 8, 24, 48, 72, and 96 hr. 4-HPR was included as a positive control and 10 μM completely eliminates cell growth at 48 hr, consistent with published results.⁴⁶

Figure 3. The effect of S-(4-methylthiobenzoyl)thioglycolate 57 and its ethyl ester 70 on the growth of human prostate DU 145 cells.

Cells were grown to 50,000 cells per well and then treated with the indicated concentration of S-(4-methylthiobenzoyl)thioglycolate or S-(4-methylthiobenzoyl)thioglycolate ethyl ester. Cell growth was measured at 8, 24, 48, 72, and 96 hr.

3. Discussion

3.1. Structure-function relationships towards the design of PHM inhibitors

The in silico docking studies show that 72 of the 79 compounds (91%) dock in the PHM active site and exhibit a relatively narrow range of Glide emodel and gscore values (Table S1, supplementary material). For the remaining 7 compounds, the Glide emodel and gscore values are still within a factor of 2 of the range for other compounds. The docking results indicate that the orientation of the substrates and inhibitors within the active site of reduced PHM is similar for 79 docked compounds included in this study. This means that a comparison of their respective binding affinities will provide valuable insights about structural determinants important for the design of tight-binding PHM inhibitors.

Note that 5 of the 7 “out of range” compounds do not terminate in a glycine moiety suggesting that there are likely (perhaps subtle) differences in active site positioning for the compounds that do not terminate in a glycine.

Previous work had shown that peptide substrates with a penultimate hydrophobic amino acid exhibited the highest V/K for oxidation.²⁰ The PHM X-ray structure containing bound N-acetyl-3,5-diiodotyrosylglycine showed that the diiodotyrosyl moiety was located in a hydrophobic pocket consisting of Asn-316 and Tyr-318.^25b Modeling studies carried out in our laboratory using Glide point toward a more extensive hydrophobic pocket within the PHM active site consisting also of Met-208, Leu-206, Ile-306, and Met-314.^¶ We purposely included a hydrophobic moiety in virtually all of the substrates and inhibitors included in this study to take advantage of the known preference exhibited by PHM for hydrophobic substrates and to take advantage of the hydrophobic pocket within the PHM active site.

Incorporation of a sulfur atom at any position in the compound increases binding affinity. The magnitude of the increase varies considerably, from a Δ(ΔG) of −0.15 kcal/mol for the 35/29 pair to ~−6 kcal/mol for the 52/59 pair (Table 6). Thiols and the -CS-S- functionality are best for increasing affinity to PHM, with Δ(ΔG) of ~−2.5 kcal/mol for thiols and −2 to −5 kcal/mol for -CS-S- (Table 7). The increase in potency due to the incorporation of sulfur atom(s) in the inhibitor is likely due to: (a) abstraction of the copper from PHM yielding inactive copper-free enzyme, (b) coordination of PHM-bound copper to form the PHM·Cu·inhibitor complex, or (c) a combination of the chelation and coordination effects. Research to develop inhibitors for the related enzyme, DβM, reached similar conclusions. Sulfur-containing compounds lead to relatively tight-binding DβM inhibitors, those exhibiting the lowest K_i values generally possessed a thiol moiety.^31c,31d Furthermore, inhibition of DβM by the sulfur-containing inhibitors could not be attributed only to chelation of enzyme-bound copper to yield inactive apo-DβM.^31b,36

Table 6.

Sulfur Atom Effect on PHM Affinity

Table 7.

Replacement of α-methylene with a -NH- spacer, R-CH₂-CO- vs. R-NH-CO-, decreases binding affinity. The magnitude of this effect is relatively small, with an average ratio of K_CH2/K_NH of ~0.5 yielding an average Δ(ΔG) of approximately −0.6 kcal/mol (Table 8). The decrease in affinity most likely results from an unfavorable interaction between the unpaired electrons of the nitrogen and the hydrophobic amino acids of the PHM active site.^25b

Table 8.

3.2. Effect of the PHM inhibitors on cultured prostate cancer cells

Two of our inhibitors, S-(2-phenylthioacetyl)thioglycolate 60 and S-(4-methylthiobenzoyl)thioglycolate 57, were antiproliferative against cultured human prostate carcinoma cells; however, these compounds were 60–300-fold less potent than their respective K_i,s values in decreasing cell viability by 50%. Similarly, Jeng et al.³⁷ reported that their PHM inhibitors were ~200-fold less potent relative to their in vitro IC₅₀ values in inhibiting the PHM-dependent formation of an α–amidated peptide, substance P, in cultured rat dorsal root ganglion cells. While some of the discrepancy between the in vitro K_i,s (or IC₅₀) values and the potency in cellular assays is likely a result of limited cellular availability, other factors such as the potential partial activity of the amidated peptide precursors,⁴ increased expression of PHM, or other non-PHM targets for these molecules could decrease in vivo potency. These data combined with similar results from Jeng et al.³⁷ and concerns about toxicity have led to questions about PHM being a viable anticancer target. However, the development of molecular “zip-codes” to specifically target a drug to its site of action could render an anti-PHM drug useful against such difficult-to-treat cancers as small-cell lung cancer,³⁸ prostate cancer,³⁹ or pancreatic cancer.⁴⁰ The structure-activity data reported here will prove useful towards the design of a tight-binding anti-PHM drug.

3.3. PHM inhibitors as novel insecticides

Most bioactive insect peptides possess a C-terminal amide² and are dependent on PHM for their biosynthesis. While there are reports of α-amidated peptides from ginseng root41 and alfalfa,⁴² we could not detect PHM activity in cell homogenates from freshly harvested alfalfa, identify a protein by Western analysis from alfalfa extracts using mammalian anti-PHM antibodies, nor identify a gene in the Arabidopsis genome that showed significant homology to human or insect PAM (Carpenter & Merkler, unpublished). These data strongly suggest that PHM does not exist in plants and that plant α-amidated peptides, if such molecules do exist, are likely produced via a non-PHM-dependent pathway. Thus, a PHM-targeted insecticide could prove relatively non-toxic to plants.

The catalytic cores of rat and Drosophila PHM show 41% sequence identity and 52% sequence similarity.^10b The sequence differences between mammalian and insect PHM coupled with the differences in K_i,s values between rat and B. germanica PHM (Table 5) strongly suggest insect specific-PHM inhibitors can be developed and could prove useful as alternative insecticides. While toxicity of an anti-PHM compound is a concern in the design of human drug, insect toxicity would be the goal of a PHM-directed insecticide.

4. Conclusion

We report here that relatively simple, substrate-like compounds can bind to mammalian PHM with K_i,s values <1 μM. A more complete understanding of the PHM mechanism should yield transition-state information leading to transition-state mimics that would exhibit better affinity for PHM. The structure-activity data reported here and elsewhere^20–24 will prove invaluable to the design of the tight-binding transition state analogs. Specific PHM-directed compounds could prove useful for the treatment of human disease, particularly certain forms of difficult-to-treat cancers, and for the development of novel insecticides.

5. Experimental Section

5.1. Materials

Bovine catalase, sodium ascorbate, MES, hippuric acid 1, aceturic acid 2, tBOC-Gly 5, 2-methylhippuric acid 6, 4-aminohippuric acid 7, N-(2-furoyl)glycine 8, 4-methylhippuric acid 11, 3-chlorohippuric acid 14, 3-methylhippuric acid 17, 4-bromohippuric acid 18, N-(2-thienylcarbonyl)glycine 19, 4-methoxyhippuric acid 20, 2-hydroxyhippuric acid 22, 4-nitrohippuric acid 23, 3-(2-furyl)acryloylglycine 26, 2-propylmercaptoacetylglycine 29, N-[(2-phenylcyclopropyl)carbonyl]glycine 30, cinnamoylglycine 40, acetyl-D-alanine 51, 6-O-palmitoyl-L-ascorbic acid 63, S-(thiobenzoyl)thioglycolic acid 64, glutathione ethyl ester 67, salicylhydroxamic acid 71, O-benzamidoglycolic acid 73, Pro-Leu-Gly hydroxamic acid 74, (S)-N-CBZ-4-amino-2-hydroxybutyric acid 75, 3-benzoylpropionic acid 77, (CBZ-hydrazido)glycine 78, (nicotinamidomethyl)phosphonic acid 80, and (R,S)-α-hydroxyhippuric acid were from Sigma-Aldrich Co.; 2-aminohippuric acid 9, 4-hydroxyhippuric acid 12, L-pyroglutamyl-Gly 25, acetyl-Gly-Gly 28, tBOC-Gly-Gly 36, hippuryl-Gly-Gly 39, hippuryl-Gly 49, and benzoyl-D-alanine 51, were from Bachem Bioscience Inc.; 2,6-difluorohippuric acid 3, 4-trifluoromethylhippuric acid 13, 4-chlorohippuric acid 21, phenylhydantoic acid 38, 2-pyridylmercaptoacetylglycine 47, and phenylthioacetylglycine 48 were from Maybridge Chemicals; N-dansyl-Tyr-Gly and 2-iodohippuric acid 4 were from Fluka Laboratory Chemicals; 3-indolylacetylglycine 24, CBZ-glycine 33, chloroacetyl-Gly-Gly 34, and CBZ-D-alanine 53 were from Research Organics Inc.; nicotinuric acid 16, phenaceturic acid 42, and 4-nitrobenzoyl-Gly-Gly 44 were from TCI America; S-phenylmercaptoacetic acid 72 was from Pfaltz and Bauer Rare and Fine Chemicals; N-phenylthiohydantoic acid 69 was from Eastern Chemical Corp.; N-benzylglycine 79 was from Indofine Chemical Co.; and N-[(benzylmercapto)carbonyl]glycine 32 was from SPECS. N-Dansyl-Tyr-Val-D-Ala^6e and S-(thiobenzoyl)-4-mercapto-4-cyanopentanoic acid 58⁴³ were synthesized as described. Human prostate DU 145 cells were purchased from the American Type Culture Collection. Adult German cockroaches (Blatella germanica) were purchased in lots of 100 from Carolina Biological Supply Co.

5.2. Enzymes

Recombinant type A rat medullary thyroid carcinoma PAM was produced and purified as described⁴⁴ and was a gift from Unigene Laboratories, Inc. (Faifield, NJ, see www.unigene.com). The rat PAM used in these studies had a specific activity ≥3.0 μmoles of O₂ consumed/min/mg at 37 °C under the following conditions: 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1% (v/v) ethanol, 0.001% (v/v) Triton X 100, 10 μg/mL bovine catalase, 1.0 μM Cu(NO₃)₂, 5.0 mM sodium ascorbate, and 11.0 mM aceturic acid.

Partially purified PAM from B. germanica was obtained by first homogenizing one hundred cockroaches (typical combined mass of 5–7 g) at 4 °C in 150 mL of 20 mM TES/NaOH pH 7.4, 10 mM D-mannitol, 0.3 mg/ml PMSF, 2 μg/ml leupeptin, and 16 μg/ml benzamidine (Buffer C). The resulting homogenate was filtered through cheese cloth, frozen and thawed three times using a dry ice-methanol bath, and then centrifuged for 20 min at 4,000 × g to remove cellular debris. The supernatant was brought to 30% of saturation by the slow addition of solid ammonium sulfate, precipitated proteins collected by centrifugation, and discarded. Solid ammonium sulfate was added to the supernatant to bring the solution to 50% saturation and the precipitated proteins again collected by centrifugation. The pellet was resuspended in Buffer C and then stored frozen at −80 °C. B. germanica PAM used in these studies had a specific activity ≥ 10 nmoles of N-dansyl-Tyr-Val-NH₂ produced/min/mg at 37 °C under the following conditions: 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1% (v/v) ethanol, 0.001% (v/v) Triton X 100, 58 μg/ml bovine catalase, 1.6 μM Cu(NO₃)₂, 10 mM sodium ascorbate, and 10.0 μM N-dansyl-Tyr-Val-Gly. The PAL specific activity of the B. germanica preparation was 2–3 nmoles of glyoxylate produced/min/mg at 37 °C under the following conditions: 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1% (v/v) ethanol, 0.001% (v/v) Triton X-100, and 2.0 mM (R,S)-α-hydroxyhippuric acid.

5.3. Growth and treatment of the human prostate DU 145 cells

The DU 145 prostate cancer cells were grown in Eagle’s minimum essential medium (EMEM) with Earle’s Salts (CaCl₂, KCl, MgSO₄, and Na₂HPO₄) and 2 mM glutamine. The growth media also contained 1.0 mM sodium pyruvate, 0.1 mM amino acids (contains all 20 amino acids), 1.5 g/L sodium bicarbonate, 10% FBS, 10,000 U/mL penicillin, and 10,000 μg/mL of streptomycin.⁴⁵ For control experiments, 10 μM 4-HPR, a compound known to be antiproliferative for DU 145 cells,⁴⁶ was also added to the growth media.

DU 145 cells were grown to 80% confluency, trypsinized, counted, and diluted to give 50,000 cells per well. The cells were allowed to adhere to the bottom of the wells for 24 hours. The media was then replaced with and without the desired inhibitor added. Cells were then counted at 8, 24, 48, 72, and 96 hours. At each time point, the media was removed and the cells washed twice with PBS. Trypsin/EDTA was added to detach cells. Media (2 mL) was added to each well, mixed, and removed. A sample from each well was taken out and Trypan blue and HBS were added. The viable and non-viable cells were counted on a hemacytometer. Viable cells appeared clear and non-viable cells appeared blue.⁴⁷

5.4. Determination of K_M,app, V_M,app, and (V/K)_app for the rat bifunctional PAM substrates

Reactions at 37.0 ± 0.1 °C were initiated by the addition of 15–50 μg of rat PAM into 2.4 mL of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1% (v/v) ethanol, 0.001% (v/v) Triton X-100, 10 μg/mL bovine catalase, 1.0 μM Cu(NO₃)₂, 5.0 mM sodium ascorbate, and the oxidizable substrate (generally 0.3 K_M,app to 5.0 K_M,app). Initial rates were measured by following the PAM-dependent consumption of O₂ using a Yellow Springs Instrument Model 53 oxygen monitor. V_M,app values were normalized to controls performed at 11.0 mM aceturic acid. Ethanol was added to protect the catalase against ascorbate-mediated inactivation and Triton X-100 was included to prevent nonspecific absorption of PAM to the sides of the oxygen monitor chambers.

5.5. Bifunctional rat PAM inhibition assays

The inhibition of rat PAM was determined by either measuring the inhibition of O₂ consumption using aceturic acid as an oxidizable substrate or the inhibition of N-dansyl-Tyr-Val-Gly amidation. Measuring O₂ consumption is more convenient, but is less sensitive and requires relatively high amounts of enzyme and inhibitor. Measuring N-dansyl-Tyr-Val-Gly amidation is a more sensitive assay for PAM activity and, thus, is most useful when enzyme and/or inhibitor are limiting.

Assays to define the inhibition of O₂ consumption were initiated by the addition of 15–50 μg of rat PAM to a solution composed of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v) ethanol, 0.001 % (v/v) Triton X-100, 10 μg/mL bovine catalase, 1.0 μM Cu(NO₃)₂, 6.0 mM sodium ascorbate, 8.0 mM aceturic acid, and a range of inhibitor concentrations (generally 0.5 K_i,s to 10 K_i,s). For compounds of lower aqueous solubility, the reaction solution contained 2.5 % (v/v) DMSO. A few experiments employed a lower substrate concentration, 5.0 mM aceturic acid, to better define the inhibition for the compounds that exhibited relatively low affinity to PAM. The final volume of the reaction solution after the addition of enzyme was 2.4 mL and the reactions were maintained at 37.0 ± 0.1 °C.

Assays for the inhibition of N-dansyl-Tyr-Val-Gly amidation were initiated by the addition of 50–100 ng of rat PAM to a solution composed of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0 % (v/v) ethanol, 0.001 % (v/v) Triton X-100, 10 μg/mL bovine catalase, 1.0 μM Cu(NO₃)₂, 6.0 mM sodium ascorbate, 8 μM N-dansyl-Try-Val-Gly, and a range of inhibitor concentrations (generally 0.5 K_i,s to 10 K_i,s). For compounds of lower aqueous solubility, the reaction solutions contained 2.5% (v/v) DMSO. A few experiments employed a lower substrate concentration, 5.0 μM N-dansyl-Try-Val-Gly, to better define the inhibition for the compounds that exhibited relatively low affinity to PAM. The final volume of the reaction solutions after the addition of enzyme was 0.5 mL and the reactions were maintained at 37.0 ± 0.1 °C throughout the course of the experiment. At the desired time, an aliquot was removed from the reaction solution and added to a vial containing one-fifth volume of 6% (v/v) trifluoroacetic acid to terminate the reaction. The acidified aliquots were assayed for the percent conversion to N-dansyl-Try-Val-NH₂ by reverse phase HPLC as described by Jones et al.⁴⁸

5.6. B. germanica PHM Inhibition Assays

Assays to define the inhibition of N-dansyl-Tyr-Val-Gly amidation were initiated by the addition of 70–200 μg of partially purified B. germanica PAM to a solution composed of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0 % (v/v) ethanol, 0.001 % (v/v) Triton X-100, 58 μg/mL bovine catalase, 1.6 μM Cu(NO₃)₂, 8.0 mM sodium ascorbate, 10 μM N-dansyl-Try-Val-Gly, and a range of inhibitor concentrations (generally 0.5 K_i,s to 10 K_i,s). The final volume of the reaction solution after the addition of enzyme was 0.5 mL and the reactions were maintained at 37.0 ± 0.1 °C throughout the course of the experiment. At the desired time, an aliquot was removed from the reaction solution and added to a vial containing one-fifth volume of 6% (v/v) TFA acid to terminate the reaction. The acidified aliquots were assayed for the percent conversion to N-dansyl-Try-Val-NH₂ by reverse phase HPLC as described by Jones et al.⁴⁸

5.7. Inhibition of PAL activity (carbinolamide dealkylation)

Dealkylation reactions at 37.0 ± 0.1 °C were initiated by the addition of 1.5–3.0 μg rat PAM into 0.3 mL of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1% (v/v) ethanol, 0.001% (v/v) Triton X-100, 2.0 mM (R,S)-α-hydroxyhippuric acid, and 0 or 750 μM inhibitor. A control lacking enzyme was included to measure both the contaminating glyoxylate in the (R,S)-α-hydroxyhippuric acid and any non-enzymatic carbinolamide dealkylation. The reactions were terminated after 20 min. at 37.0 ± 0.1 °C by the addition of 60 μL of 6% (v/v) TFA acid. Dealkylation activity of the partially purified B. germanica preparation was measured similarly except that the assays were initiated by the addition of 54 μg protein into a 0.6 mL reaction volume and the reactions were incubated for 6 hrs at 37.0 ± 0.1 °C before an aliquot was removed for glyoxylate quantification.

The concentration of glyoxylate in the acid quenched samples was determined spectrophotometrically via the method of Christman et al.⁴⁹ as modified by Katopodis and May.²⁴ Standard curves of [glyoxylate] vs. A₅₂₀ were constructed in the appropriate buffers using a glyoxylate solution that had been calibrated by measuring the glyoxylate-dependent oxidation of NADH (Δε₃₄₀ = 6.22 × 10³ M⁻¹ cm⁻¹) as catalyzed by lactate dehydrogenase.

The single time point assays for glyoxylate served only to screen for PAL inhibition from the set of compounds under study. Only one compound, S-(phenylthiocarbamoyl)thioglycolic acid 61, showed appreciable inhibition (~40%) of the rat PAM dealkylation activity from the initial screen. To more fully characterize this inhibition, 8.3 μg of rat PAM was added to 1.0 mL of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1% (v/v) ethanol, 0.001% (v/v) Triton X-100, 2.0 mM (R,S)-α-hydroxyhippuric acid, and 0–6.0 mM S-(phenylthiocarbamoyl)thioglycolic acid 61. At 5 min intervals, a 0.15 mL aliquot was removed, and added to a vial containing 30 μL of 6 % (v/v) TFA to terminate the reaction. Glyoxylate was quantified in the acidified samples as described above.

5.8. Analysis of initial rate kinetic and inhibition data

Initial velocities, v, obtained as a function of the initial substrate concentrations, [S], were fit to eq. 1 using KaleidaGraph^™.

$$\text{v}=({\text{V}}_{\text{M},\text{app}}[\text{S}])/({\text{K}}_{\text{M},\text{app}}+[\text{S}])$$ (1)

The K_M,app is the apparent Michaelis constant for S at constant fixed initial concentrations of the co-substrates (5.0 mM sodium ascorbate and 200 μM O₂ for PAM). The V_M,app is the apparent catalytic rate at saturating [S] under the conditions of the experiment.

Values for K_i,s were obtained by a KaleidaGraph^™ fit of the data for the dependence of the initial velocity, v, on the concentration of inhibitor, [I], at one fixed, initial substrate concentration, [S] to eq. 2. We have assumed, in using eq. 2, that the inhibitors are competitive with the oxidizable substrate, aceturic acid or N-dansyl-Tyr-Val-Gly.

$$\text{v}=({\text{V}}_{\text{M},\text{app}}[\text{S}])/\{{\text{K}}_{\text{M},\text{app}}+{\text{K}}_{\text{M},\text{app}}([\text{I}]/{\text{K}}_{\text{i},\text{s}})+[\text{S}]\}$$ (2)

The IC₅₀ values for the inhibition of N-dansyl-Tyr-Val-Gly amidation by 6-O-palmitoyl-L-ascorbate and the inhibition of (R,S)-α-hydroxyhippuric acid dealkylation by S-(phenylthiocarbamoyl)thioglycolic acid 61 were calculated as described by Cortés et al.⁵⁰

5.9. In silico docking

The crystal structure for reduced peptidylglycine α-hydroxylating monooxygenase (PHM) was obtained from the Protein Data Bank (http://www.rcsb.org/pdb/, 1SDW).^25c All co-crystallized species determined to be redundant for ligand binding were removed (nickel, water, glycerol, and substrate). Formal charges for enzyme-bound copper ions and bond orders were corrected, and hydrogens were added using Maestro (www.schrodinger.com). Further receptor refinements were carried out utilizing ProteinPrep from within Maestro. Glide and Q-site from the FirstDiscovery 3.0 suite (www.schrodinger.com) were used for quantum polarized ligand docking (QPLD) to generate highly accurate ligand binding modes.⁵¹

5.10. Synthesis of isocaproylglycine 35

To a stirred, chilled solution of isocaproyl chloride (1.35 g, 10 mmole) in 10 mL of dichloromethane was added glycine methyl ester hydrochloride (1.25 g, 10 mmole), followed by the dropwise addition of triethylamine (2.2 g, 22 mmole). The mixture was stirred at room temperature overnight and then treated with 20 mL of 1.0 M HCl. The dichloromethane layer was washed with a sodium bicarbonate solution, dried with sodium sulfate, and stripped of solvent. The yield of methyl isocaproylglyincate was 1.32 g (70%) and the structure confirmed by ¹H NMR. A portion of the crude ester (0.93 g, 5 mmole) was stirred with 1.0 M NaOH (5.5 mL) until all dissolved (25 min), chilled, and acidified with 6.0 M HCl (2 mL). The oil, which separated slowly, crystallized. Recrystallized twice from ether:pentane (2:1), isocaproylglycine (0.4 g, 50%) had mp 91–92, ¹H NMR (CDCl₃): δ 0.86–0.88 (m, 6H), 1.47–1.57 (m, 6H), 2.27 (t, J=8.0 Hz, 2H), 4.01–4.03 (m, 2H), 6.51–6.54 (m, 1H), and 8.03–8.24 (br. s., 1H).

5.11. General synthesis of substrates and inhibitors

Glycine conjugate 27 (N-(α-methylhydrocinnamoyl)glycine) was prepared as described by McIntyre et al.^23d Glycine conjugates 10, 15, 31, 43, 45, 46, and 50 were prepared similarly. Acylation of cysteine and serine requires careful pH control,⁵² otherwise considerable racemization will occur.⁵³ At pH 9.5, unracemized 68 and 76 were obtained satisfactorily. Thioacylglycine 48 (phenylthioacetylglycine) was prepared using a literature procedure,⁵⁴ and 41 and 59⁵⁵ were prepared in a similar fashion. S-(Thioacyl)thioglycolic acids, 55,⁵⁶ 57,⁵⁷ 60,^54,57 62,⁵⁷ and 66,⁵⁸ were made using the literature procedures cited here. The ethyl ester of 57, S-(4-methylthiobenzoyl)thioglycolic acid ethyl ester 70,⁵⁹ was an adventitious by-product when the acid was prepared in ethanol using thioacetic acid in place of H₂S using the procedure of Jensen and Pedersen.⁵⁷ Thiourea 69⁶⁰ (N-phenylthiohydantoic acid) and dithiocarbamates 56 and 61 were prepared as described,⁶¹ as was carbamate 65⁶² (O-(phenylcarbamoyl)glycolic acid). Several of the compounds detailed here are mentioned in the prior literature as synthons or as isolates from biological systems, but physical and spectroscopic data were not reported. We detail this information in the next section: Physical and spectroscopic properties.

5.12. Physical and spectroscopic properties

The solvent used in obtaining the NMR spectra was CDCl₃, unless otherwise noted. Satisfactory elemental analysis, C, H, N, and S, was obtained for all compounds.

10: ¹H NMR δ 1.58 (t, J=7.6 Hz, 3H), 2.60 (q, J=7.6 Hz, 2H), 4.10 (d, J=4.7 Hz, 2H), 7.05 (br. s, 1H), 7.16 (d, J=7.7 Hz, 2H), and 7.67 (d, J=7.7 Hz, 2H).

31: mp 118 (Davies et al.,⁶³ mp 116–118).

41: mp 125–126. ¹H NMR δ 2.86 (t, J=7.2 Hz, 2H), 2.97 (t, J=7.0 Hz, 2H), 4.19 (d, J=4.8 Hz, 2H), 7.06–7.15 (m, 5H), and 9.37 (s, 1H).

48: mp 142 (Fry,⁵³ mp 141–142).

50: mp 88–90.

55: mp 66–68 (Leon,⁵⁶ mp 64.5–66).

56: mp 157–158 (Cherbuliez et al.,^61b mp 159–160). ¹H NMR (CDCl₃-[D₆]-DMSO) δ 2.64 (t, J=6.9 Hz, 2H), 3.41 (t, J=6.9 Hz, 2H), 7.09–7.49 (m, 5H), and 11.00 (s, 1H). ¹³C NMR δ 30.16, 34.00, 124.28, 126.56, 128.71, 139.35, and 173.11.

57: mp 116–117 (Jensen and Pedersen,⁵⁷ mp 118–119). ¹H NMR δ 2.40 (s, 3H), 4.24 (s, 2H), 7.20 (d, J=8.1 Hz, 2H), 7.97 (d, J=8.2 Hz, 2H), and 10.21 (br. s, 1H). ¹³C NMR δ 21.57, 38.58, 127.09, 129.13, 141.51, 144.10, 173.79, 243.

59: mp 124 (Barrett,⁵⁵ mp 124).

60: mp 55^||. ¹H NMR δ 4.04 (s, 2H), 4.33 (s, 2H), 7.21–7.32 (m, 5H), and 10.3 (br. s, 1H). ¹³C NMR δ 38.50, 57.27, 127.46, 128.64, 129.11, 136.30, 173.24, 233.10.

61: mp 145 (fast), 192–193 (slow) (Cherbuliez et al.,^61a compound cyclizes to a thioxothiazoldine, mp 193). ¹H NMR (CDCl₃-[D₆]-DMSO) δ 3.96 (s, 2H), 7.09–7.45 (m, 5H), 9.28 (br. s., 1H), and 10.79 (s, 1H). ¹³C NMR δ 39.54, 124.03, 128.31, 128.73, 129.40, and 170.68.

62: mp 88–90 (Jensen and Pedersen,⁵⁷ mp 92–93). ¹H NMR δ 2.95–3.15 (m, 4H), 3.87 (s, 2H), 6.7–7.21 (m, 5H), and 10.1 (s, 1H).

65: mp 141–143 (Fischer and Fischer,⁶² mp 142–143). ¹H NMR (CDCl₃-[D₆]-DMSO) δ 4.51 (s, 2H), 6.91 (t, J=7.3 Hz, 1H), 7.17 (t, J=7.7 Hz, 2H), 7.42 (d, J=8.0 Hz, 2H), and 9.62 (s, 1H). ¹³C NMR δ 60.73, 118.62, 122.79, 128.79, 139.05, 153.18, and 170.13.

68: changes form at 173, melts at 182 (according to Fischer and Fischer,⁶² 68 shrinks at 173 and melts at 179–182).

69: mp 142–144 (fast), >200 (slow, compound cyclizes) (Dash et al.,⁶⁰ mp 141). ¹H NMR (CDCl₃-[D₆]-DMSO) δ 4.21 (d, J=5.0 Hz, 2H), 7.05 (t, J=7.3 Hz, 2H), 7.21–7.42 (m, 5H), 7.62 (t, J=4.7 Hz, 1H), and 9.67 (s, 1H). ¹³C NMR δ 46.11, 123.46, 124.72, 128.85, 139.15, 171.40, 70: mp 33–35, bp 185–190/1mm. ¹H NMR δ 1.29 (t, J=7.1 Hz, 3H), 2.37 (s, 3H), 4.18–4.26 (m, 4H), 7.18 (d, J=8.0 Hz, 2H), and 7.96 (d, J=7.9 Hz, 2H).

76: mp 150 (Kameda et al.,⁶⁴ mp 149–150).

Abbreviations

tBOC

tert-butyloxycarbonyl

CBZ

carbobenzyloxy

Dansyl

[5-(dimethylamino)-1-naphthalenyl]sulfonyl

DβH

dopamine β-monooxygenase

4-HPR

N-(4-hydroxyphenol)retinamide

MES

4-morpholineethanesulfonic acid

PAL

monofunctional peptidylamidoglycolate lyase

PAM

bifunctional peptidylglycine α-amidating monooxygenase

PHM

monofunctional peptidylglycine α-hydroxylating monooxygenase

SAR

structure-activity relationship