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Substrate-based inhibitors of peptidylglycine á-amidating monooxygenase (PAM) as anti-proliferative drugs for cancer

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Substrate-based inhibitors of peptidylglycine á-amidating monooxygenase (PAM) as anti-proliferative drugs for cancer
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Chew, Geoffrey H
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enzyme
prostate
inhibition
4-hpr
kinetics
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Abstract:
ABSTRACT: C-Terminal glycine-extended prohormones are enzymatically converted to á-amidated peptides, by peptidylglycine á-amidating monooxygenase (PAM). PAM is a bifunctional enzyme with two catalytic domains: peptidylglycine á -hydroxylating monooxygenase (PHM) and peptidylglycine peptidylglycineaminoglycolate lyase (PAL). PAM has a significant role in the proliferation of androgen-independent prostate cancer. Thus, the inhibition of PAM could halt cancer growth. Hippurate and hippurate analogs were used as lead compounds for developing inhibitors for PAM. The hippurate analogs exhibiting the highest affinity to PAM (lowest inhibition constant) did inhibit the growth of human androgen-independent prostate cancer DU 145 cells.
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Thesis (M.S.)--University of South Florida, 2003.
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Includes bibliographical references.
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by Geoffrey H Chew.
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Substrate Based Inhibitors of Peptidylglycine Amidating Monooxygenase (PAM) as Anti Proliferative Drugs for Cancer by Geoffrey H. Chew A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: David Merkler, Ph.D. Randy Larsen, Ph.D. Gloria Ferreira, Ph.D. Date of Approval: November 16, 2003 Keywords: enzyme, prostate, inhibition, 4 hpr, kinetics Copyright 2003, Geoffrey H. Chew

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i Table of Contents List of Tables List of Figures List of Graphs Abstract Chapter One Literature Review Peptidylglycine amidating monooxygenase Prostate cancer C onclusion Chapter Two Novel Compounds for PAM Introduction Enzyme inhibition N Benzoylgly cine (hippuric acid) as lead compound M ethods to assay for PAM activity Experimental Procedures Materials High performance liquid chromatography HPLC separation of dansylated compounds Determination of initial rates of N dansyl Try Val NH 2 production as fixed N dansyl Try Val Gly concentration as a function of inhibitor concentration (Dixon analysis) Determination of K M and V MAX values for N dansyl Tyr Val Gly as a function of inhibitor concentration Determination of initial rates by oxygen consumption Copper chelating experiment Molecular Modeling R esults and Discussion PAM substrates and inhibitors Copper chelating experiment Determination of K M and V MAX values for N dansyl Tyr Val Gly as a function of inhibitor concentration iii iv v vi 1 1 5 8 9 9 9 11 11 13 13 14 14 15 16 16 17 17 18 18 27 27

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ii Conclusion Chapter Three Prostate Cancer Testing Introduction Experimental Procedures Materials Cells and cell culture Anti proliferation assay of S (4 methylthiobenzoyl)thioglycolic acid Anti proliferation assay of S (phenylthioacetyl)thioglycolic acid R esults and Discussion Anti proliferation assay of S (4 m ethylthiobenzoyl)thioglycolic acid Anti proliferation assay of S (phenylthioacetyl)thioglycolic acid Conclusion Referenc es 29 31 31 32 32 32 33 33 34 34 35 37 39

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iii List of Tables Table 1. Kinetic parameters for N dansyl (Gly) 4 X Gly amidation Table 2. List of PAM inhibitors Table 3. List of PAM substrates Table 4. Summary of bond energies Table 5. Comparison of inhibitor and substrate bond energies and binding constants Table 6. Comparison of substrates between the two assays 5 19 20 23 24 26

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iv List of Figures Figure 1. The PAM catalyzed reaction Figure 2. 3D structure of PHM with bound substrate Figure 3. Sequence comparison of PHM and DBM Figure 4. Structure of 4 HPR Figure 5. Diagram of three main types of inhibition Figure 6. Diagram of irreversible inhibition Figure 7. Sites of different binding energies Figure 8. Formula for calculating free energies f or the complete docking of a compound into the active site of PHM 2 3 4 7 10 10 21 22

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v List of Graphs Graph 1. PAM activity in different prostate cancers Graph 2. PAM growth curve with AM Graph 3. PAM growth curve with 4 HPR Graph 4. Copper chelating experiment with EDTA Graph 5. Full kinetic study on S (3 phenyl thiopropionyl)thioglycolic acid Graph 6. Full kinetic study on octanoyl glycine Graph 7. Anti proliferative assay of S (4 methylthiobenzoyl)thioglycolic a cid Graph 8. Anti proliferative assay of S (phenylthioacetyl)thioglycolic acid 6 6 8 28 29 30 35 36

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vi Substrate Based Inhibitors of Peptidylglycine Amidating Monooxygenase (PAM) as Anti Proliferative Drugs for Cancer Geoffrey H. Chew ABSTRACT C Terminal glycine extended prohormones are enzymatically converted to amidated peptides, by peptidylglycine amidating monooxygenase (PAM). PAM is a bifunctional enzyme with two catalytic domains: peptidylglycine hydro xylating monooxygenase (PHM) and peptidylglycine p eptidylglycineaminoglycolate lyase (PAL). PAM has a significant role in the proliferation of androgen independent prostate cancer. Thus, the inhibition of PAM could halt cancer growth. Hippurate and h ippurate analogs were used as lead compounds for developing inhibitors for PAM. The hippurate analogs exhibiting the highest affinity to PAM (lowest inhibition constant) did inhibit the growth of human androgen independent prostate cancer DU 145 cells.

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1 Chapter One Literature Review Peptidylglycine amidating monooxygenase Approximately 50% of all mammalian hormones are amidated, and play important roles in biological systems. Many hormones, such as human growth hormone releasing factor (GRF), are only active in the amidated form (Merkler 1994). C Terminal glycine extended prohormones are enzymatically converted to amidated peptides, by peptidylglycine amidating monooxygenase (PAM). PAM is the only known enzyme that performs this chemistr y (Eipper 1988). PAM enables the oxidative cleavage of inactive C terminal glycine extended prohormones to active amidated peptides and glyoxylate. PAM is a bifunctional enzyme with two catalytic domains: peptidylglycine hydroxylating monooxygenase (P HM) and peptidylamidoglycolate lyase (PAL) (Merkler, 1995). PHM catalyzes the O 2 copper and ascorbate dependent insertion of oxygen from an O 2 molecule at the C a of the terminal glycyl residue to form a carbinolamide while PAL catalyzes the zinc dep endent dealkylation of the carbinolamide to the a amidated peptide and glyoxylate ( Jaron, 2002) (Figure 1).

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2 P e p t i d e 1 5 N H 1 4 1 4 C O O H O O 2 H 2 O 2 A s c o r b a t e 2 S e m i d e h y d r o a s c o r b a t e P H M P e p t i d e 1 5 N H 1 4 C H 1 4 C O O H O O H 2 C u ( I I ) Z n ( I I ) A L 4 G y o y l t e e p i d 1 5 H 2 Figure 1 The peptide amidation reaction as catalyzed by PAM. PAM is a bifunctional protein comprised of two dis tinct catalytic entities: peptidylglycine hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL). Labeled nitrogens and carbons were used in experiments to determine products of the reaction. Early work performed using bifunctional PAM defined essential characteristics of the peptide amidation reaction. Incubation of D Tyr Val [ glycyl 15 N] Gly with bifunctional PAM yields D Tyr Val [ 15 N] NH 2 which demonstrates the glycyl nitrogen is retained in the final amide (Bradbury 1982). In a similar experiment, D Tyr Val [ 14 C] Gly was used as a substrate. The labeled carbon atoms were incorporated only into glyoxylate. Eipper et al. (1983) showed that molecular oxygen, copper, and an electron donor were required for the PAM reaction to oc cur. Metal chelators completely abolished PAM activity that only could be restored upon the addition of copper -at a slight excess over the residual chelator concentration. The turnover of each glycine extended peptide to the a amidated peptide require s the input of two electrons, which can be donated from many different reductants. Ascorbic acid is the most efficient electron donor, exhibiting the highest (V MAX /K M ) app at constant peptide substrate concentration, and is likely the reductant utilized by PAM in vivo (Merkler 1992). After the discovery that PAM is bifunctional and that the C terminal a hydroxyglycine extended peptides are intermediates in the amidation reaction, it was established that O 2 copper, and ascorbate were only required for the PHM reaction. Figure 2 shows the 3D structure of PHM with a bound substrate. Note the presence o f two PHM bound copper atoms, one on either side of the binding pocket.

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3 Figure 2 3D structure of PHM with bound substrate. EPR studies performed by Freeman et al. (1993) demonstrated that PHM bound Cu (II) redox cycles from Cu (II) to Cu (I) back t o Cu (II) during catalysis. All of the copper ions before the addition of ascorbate are spin active. After the addition of ascorbate, 95% is no longer spin active, which indicated that the Cu (II) is reduced to Cu (I). After reaction with the substrate, Cu (I) reverts back to Cu (II). PHM and dopamine monooxygenase (DBM) exhibit a number of catalytic similarities. Both enzymes insert an oxygen atom from O 2 into an inactivated C H bond, have a copper stoichiometry of two copper atoms per active site, and oxidize two ascorbate molecules to semihydroascorbate per enzyme turnover. In addition to catalytic similarities, the two enzymes have similar protein sequence (Figure 3) and expression levels.

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4 PVTPLDASDFALDIRMPGVT PKESDTYFCMSMRLPVD -------EEAFVIDFK PRASM 103 P P D +DIR P V TY+C LP+ EA V + + PAMPADVQ -TMDIRAPDVLIPSTETTYWCYITELPLHFPRHHIIMYEAIVTE ---GNE 76 DTVHHMLLFGCNMPSSTGSYWF -CDEGTCTDKAN ---ILYAWARNAPPTRLPKGVGFR 157 VHHM +F C S + CD D+ N +L AWA A P+ G ALVHHMEVFQCTNESEAFPMFNGPCDSKMKPDRLNYCRHVLAAWALGAKAFYYPEEAGVP 136 VGGETGSKYFVLQVHYGDISAFRDNHKDCSGVSVHLTRVPQPLIAGMYLMMSVDT --VI 214 +G S++ L+VHY + + +D SG+ +H T +P AG+ + V T I LGSSGSSRFLRLEVHYHNPRNIQ GRRDSSGIRLHYTASLRPNEAGIMELGLVYTPLMAI 195 PPGEKVVNADISCQYKMYPM ------HVFAYRVHTHHLGKVVSGYRVRNGQWT LIGRQ 266 PP E C + M +FA ++HTH G+ V R+GQ ++ R PPQETTFVLTGYCTDRCTQMALPKSGIRIFASQLHTHLTGRKVITVLARDGQQREVVNRD 255 NPQLP QAFYPVEHPVDVTFGDILAARCVFTGEGRTEATHIGGTSSDEMCNLYIMYYME 324 N P Q +++ V V GD+L C + E RT AT G +EMC Y+ YY + NHYSPHFQEIRMLKNAVTVHQGDVLITSCTYNTENRTMATVGGFGILEEMCVNYVHYYP K 315 AKYAL 329 + L TELEL 320 Figure 3 Compared protein sequences of rat PHM and DBM using BlastP. There is 29% identity and 41% positives. Considerably less is known about the PAL reaction. Model studies indicate that carbinolamide dealkylation is base catalyzed (Bundgaard 1991). These data suggest that an enzymatic base abstracts the hydroxyl proton to facilitate conversion to the amide and glyoxylate. Another possibility is that a zinc based hydroxyl is the enzymatic base; thus, accounting for the zinc dependence of the PAL reaction ( Bell 1997 ). Using a set of glycine extended peptides of the form N dansyl (Gly) 4 X Gly, Tamburini et al. (1990) demonstrated that PAM could produce peptides terminating all 20 amino acid amides. The penultimate a mino acid does have an effect on the kinetic constants for amidation. The V MAX /K M value obtained for N dansyl (Gly) 4 Phe Gly was 1140 fold higher then that obtained for N dansyl (Gly) 4 Glu Gly. The glycine extended peptides with a sulfur containing or hydrophobic amino acid in the penultimate

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5 position exhibited the highest V MAX /K M values (Table 1). This result was critical in our development of novel sulfur containing hippurate analogs as PAM inhibitors. X K M ( m m M) V MAX (nmol/min/mg) Relative V MAX /K M X K M (mM) V MAX (nmol/min/mg) Relative V MAX /K M Phe 4 50 1140 Ala 46 6 12 Tyr 5 40 730 Asn 83 7 7.6 Met 7 23 300 Arg 200 15 6.8 Ile 20 55 250 Gln 308 17 5.0 Trp 58 58 91 Ser 196 9 4.2 Val 49 48 89 Pro 618 22 3.3 Cys 11 10 83 Lys 206 4 1.7 Leu 54 22 37 Glu 449 5 1.0 His 41 10 22 Gly <0.4 Ala 334 50 14 Asp Table 1 Kinetic parameter for N dansyl (Gly) 4 X Gly amidation Replacement of the C terminal glycine with any other D or L amino acid, with the exception of D alanine, did not support PAM catalysis. D Alanine extended peptides are relatively poor PAM substrates, exhibiting V MAX /K M values that are ~0.1% of that obtained for the corresponding glycine extended peptides. Based on this finding, hippurate and hippurate analogs were used as a lead compounds for developing inhibitors for PAM. The role of PAM in prostate cancer Prostate cancer is the second leading cause of cancer death in males today. The probability that a man will contract prostate cancer in his lifetime is reachi ng 100% (Parker 1996). There are two basic types of prostate cancer: androgen dependent and androgen independent (Bonkhoff 1993). Androgen dependent prostate cancer cells have

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6 Graph 1 PAM activity in androgen dependent (LnCaP)and androgen independen t (PC 3 and DU 145) prostate cancer cell lines. many androgen receptors. When deprived of androgen, these cells die. Androgen independent cells do not have these receptors and do not rely on androgen to proliferate (Rocchi 2001). A common androgen dep endent cell line is LnCaP, and common androgen independent cell lines are PC 3 and DU 145. Androgen dependent forms of cancer can be effectively treated by therapeutic hormone deprivation (Kyprianou 1990), but in most cases where androgen dependent prosta te cancer has been suppressed, it remerges as an androgen independent form. The pathway for this change is not known, but it is thought that PAM has a role in the proliferation of the newly formed androgen independent cancer. In androgen independent strai ns of prostate cancer, there is a 3 fold greater amount of PAM mRNA. There are also elevated PAM protein levels (Graph 1). Graph 2 Effect of AM on cell growth of DU 145 prostate cancer cells.

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7 The specific activity of the PAM found in the androgen ind ependent cell lines is also higher. Adrenomedullin (AM), a growth stimulating amidating peptide, is produced by androgen independent prostate tumors (Rocchi 2001). AM is only active in its amidated form. Graph 2 shows the growth rates of DU 145 cells that have been treated with AM. Ther e is a significant increase in cell growth when AM is added. AM does not affect androgen dependent cells. It is thought that the amount of AM that can be used in these cell lines are at their maximum, thus adding more peptide does not affect these cells (Rocchi 2001). N (4 hydroxyphenyl) retinamide (4 HPR) is a known inhibitor of DU 145 Me Me Me Me Me N H O OH E E E E Figure 4 Structure of 4 HPR. cells (Figure 4). It is thought that 4 HRP undergoes an oxidative pathway that generates reactive oxygen species. These oxygen speci es are extremely dangerous to cells, since they interrupt pathways by inactivating enzymes. The aminophenol ring and long alkyl chain are key functional groups for drug cell contact (Takahashi 2002). Aminophenols such as p methylaminophenol ( p MAP), 4 a minophenol (4 AP), and p aminoacetophenol ( p AAP) have also been shown to stop cell growth. Graph 3 shows the effect of 4 HPR and several aminophenols on the growth of DU 145 cells.

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8 Graph 3 Effects of 4 HPR on DU 145 prostate cancer cell growth. Cel ls were grown with compound for 72 hours before counting. The clear squares represent control cells, the clear triangles represent 4 HPR, the solid squares represent p MAP, the solid circles represent 4 AP, and the solid diamonds represent p AAP. Conclus ion Around 50% of all mammalian hormones have the C terminal amidated structure that PAM produces. PAM plays a critical role in the emergence of androgen independent forms of prostate cancer. The experiments in the following chapters use substrate based in hibitors targeted for PAM. The kinetic constants were experimentally detected and the most potent compounds were tested on prostate cancer cells to determine if they would halt DU 145 cell proliferation.

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9 Chapter Two Substrate Based Inhibitors for P AM INTRODUCTION Inhibitors are substances that lower the rate of catalysis of an enzyme. Inhibiting enzymes are one of the most common ways that organisms regulate their biological processes. The binding specificity of a substance towards an enzyme can be determined by studying the inhibition (Segel 1976). In many types of cancer, there are mutated enzymes that cause the spread of the disease, thus inhibiting these enzymes is key to halting cancer proliferation. Enzyme Inhibition Three types of inhibition have been described that account for the reversible binding of one inhibitor molecule, I, to one enzyme molecule, E (Figure 5). For competitive inhibition, the inhibitor and substrate, S, compete exclusively for E yielding either the catalytic ally competent ES complex or the catalytically incompetent EI complex (Figure 5a). Non competitive inhibition involves the binding of I to E and ES to producing both an EI binary complex and an ESI ternary complex. The affinity of I to E and ES is descri bed by two dissociation constants, K is and K ii respectively (Figure 5b). If K is equals K ii the inhibition is usually referred to as non competitive or pure non competitive inhibition. If K is does not equal K ii the inhibition is referred to as mixed

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10 ty pe inhibition. Uncompetitive inhibition is the binding of I only to the ES complex to generate the inactive ESI complex (Figure 5c). Segal (1976) details other inhibition types, usually involving the reversible binding of more than one inhibitor molecule per enzyme molecules. Such modes of inhibition are infrequently encountered. E + S + E S E I I E + P K s k p K i F i g u r e 5 D i a g r a m o f a ) c o m p e t i t i v e i n h i b i t i o n b ) n o n c o m p e t i t v e i n h i b i t i o n a n d c ) u n c o m p e t i t i v e i n h i b i t i o n E + S + E S + E I + S I E + P K s k p K i s I E S I a K s K i i a ) b ) E + S E S + E + P K s k p I E S I K i c ) Irreversible inhibitors are well known in the enzyme literature and, most often, involve the generation of an inactive enzyme species after the initial formation of the EI complex (Figure 6). The mechanism outlined in Figure 6 can account for mechanism based, suicide substrates, the chemical modification of active site resides, and slow binding inhibitors (k inact <<< k p ). E + I E I ( E I ) i n a c t i v e K i k i n a c t F i g u r e 6 D i a g r a m o f i r r e v e r s i b l e i n h i b i t i o n F o r s l o w b i n d i n g i n h i b i t i o n S a n d I c o m p e t e f o r E ( s i m i l a r t o c o m p e t i t i v e i n h i b i t i o n ) a n d k i n a c t < < < k p

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11 N Benzoylglycine (Hippuric Acid) as Lead Compound Tamburini et al. (1990) demonstrated that PAM preferred peptide substrates with hydrophobic or sulfur containing amino acids at the penultimate position. Hippurate is an ideal candidate as an ideal lead compound for the development of small molecules inhibitors of PAM for the following reasons: (a) hippurate is a known PAM substrate, (b) hippurate contains a hydrophobic N benzoyl group at a position analogus to the penultimate amino acid in C t erminal glycine extended peptides, (c) numerous ring substituted hippurates and hippurate analogs are commercially available, (d) two sulfur containing hippurate analogs were commercially available, and (e) targeted analogs of hippurate that are not availa ble commercially (with and without sulfur) are relatively straightforward to synthesize. Given these considerations, we have prepared and evaluated a library of substituted hippurates and hippurate analogs as substrates and inhibitors. The inhibitors with the highest affinity for PAM (lowest values of K is and K ii ) were then tested for anti proliferative behavior vs. prostate cancer cells in culture (Chapter 3). Methods to Assay for PAM Activity PAM converts C terminal glycine extended peptides to amidated peptides and glyoxylate in a reaction that consumes O 2 and reductant (Figure 1). Thus, PAM activity can be measured by assaying for (a) the consumption of O 2 (b) the consumption of reductant (c) the production of glyxoylate, and (d) the production of the amidated peptide. These different assay methods have advantages and disadvantages and all have been appropriately applied in the study of PAM. The two assay methods employed to

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12 generate the data contained within this thesis were to measure PAM dependent conversion of N dansyl Tyr Val Gly to N dansyl Tyr Val NH 2 by HPLC and to measure the PAM dependent consumption of O 2 in the presence of an oxidizable substrate. The PAM dependent conversi on of N dansyl Tyr Val Gly to N dansyl Tyr Val NH 2 has been extensively used in the Merkler laboratory (Merkler 1999). This assay is sensitive, requiring relatively small amounts of enzyme and N dansyl Tyr Val Gly, and can be carried in volumes <<1.0 ml, particularly important if the small amounts of inhibitor are available However, this method is laborious, provides only a discontinuous measure of [product] formed per unit time, and requires the availability of an HPLC equipped with an autosampler, and an inline flow through fluorescence detector. The development of new HPLC separation methods and syntheses of substrate and product standards are necessary to extend this assay method to other N dansylated (or otherwise fluorescently lableled) PAM substr ates. The PAM dependent consumption of O 2 has also been extensively employed in the Merkler laboratory ( Merkler 1999 ). This method requires only an O 2 electrode, provides a continuous measure of [substrate] consumed per unit time, and is generally useful for any oxidizable substrate. Unfortunately, this assay is less sensitive than assaying for N dansyl Tyr Val NH 2 production and requires considerable more PAM and substrate. Furthermore, this system has an inherent background rate of O 2 consumption Non fluorescent substrates that compete with N dansyl Tyr Val Gly for the PAM active site behave as competitive inhibitors (Spector 1981). The K is values obtained using non fluorescent substrates equal the K M The PAM dependent conversion of N dansyl Tyr Val Gly to N dansyl Tyr Val NH 2 is useful to determi ne K M values for non fluorescent substrates of limited availability or those that exhibit low V MAX values.

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13 (from the O 2 electrode and the presence of reductant and Cu(II) ions in the assay buffer) that renders this method difficult to use with substrates of with low V MAX values. EXPERIMENTAL PROCEDURES Materials MES buffer, N dansyl Try Val Gly, hippuric acid an d S (thiobenzoyl)thioglycolic acid were from Sigma, sodium chloride, ethanol, Triton X 100, HEPES buffer, acetonitrile, sodium acetate, sodium hydroxide, and trifluoroacetic acid were from Fisher Scientific, sodium ascorbic acid was from Research Organics, copper nitrate and isopropanol were from Acros, and catalase was from Worthington Biochemical Corporation. N Acetylglycine was from T.C.I. Oxygen monitor and electrodes were from Yellow Spring Instrument. Signal amplifier was from Oriel and assembled b y Dr. R. Larsen (University of South Florida). N Phenylhydantoic acid was from Maybridge. (D,L) Thiorphan was from BACHEM. 4 Ethylhippuric acid, N (phenylthiopropionyl)glycine, 4 propionylhippuric acid, N (6 phenylhexanoyl)glycine, N (8 phenyloctanoyl)g lycine, S (4 methylthiobenzoyl)thioglycolic acid, S (4 methylthiobenzoyl)thioglycolic acid ethyl ester, S (phenylthioacetyl)thioglycolic acid, S (N phenylthiocarbamoyl)thioglycolic acid, S (N phenylthiocarbamoyl) 3 mercaptopropionic acid, N glycolic acid p henyl urethane, lauroyl sulfanyl acetic acid and S (3 phenylthiopropionyl)thioglycolic acid were from Dr. T. C. Owen (University of South Florida). (Phenylthio)acetic acid and (2 nitrophenylthio)acetic acid were from Dr. J. Vederas (University of Alberta) N (Phenylthioacetyl)alanine and 4 cyano 4 methyl 4

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14 thiobenzoyl sulfanyl butyric acid were from Dr. A. Lowe (University of Southern Mississippi). HyperChem 7.5 was from Hypercube Inc. High performance liquid chromatography N Dansyl Try Val Gly and N dansyl Try Val NH 2 were separated using a Hewlett Packard 1100 Series liquid chromatograph equipped with a quaternary solvent delivery system, vacuum degasser, temperature controlled column compartment, and an auto injector. A Keystone Scientific Operatio ns Thermo Hypersil reverse phase C 18 column (100 mm x 4.6 mm, 5 m m particles, 120 pore) fitted with a Phenomenex C 18 Security Guard column was used for the separation. A Gilson Model 121 flurometer and a Hewlett Packard 3392A integrator monitored separa tions. Hewlett Packard ChemStation controlled the 1100 Series liquid chromatograph. Bifunctional peptidylglycine a amidating monooxygenase Chinese hamster ovary cells that secrete recombinant type A rat medullary thyroid carcinoma PAM into the culture media (Bertelsen, 1990) were grown in a Cellco Cellmax 100 hollow fiber bioreactor (Matthews, 1994). The bifunctional enzyme was purified as described by Miller et al. (1992) except that the final gel filtration step (Superdex 200) was done using 50 mM T ris pH 8.0 and 100 mM NaCl. The amidation of N dansyl Tyr Val Gly to N dansyl Tyr Val NH 2 (Jones 1988) and UV detection at 280 nm were used throughout the enzyme purification to screen column fractions.

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15 HPLC separation of dansylated compounds N Dans yl Try Val Gly and N dansyl Try Val NH 2 were separated on a reverse phase C 18 column (100 mm x 4.6 mm, 5 m m particles, 120 pore). The mobile phase for the separation consisted of 100 mM sodium acetate, 55% deionized water and 45% acetonitrile. The mobile phase was delivered at a flow rate of 1.2 ml/min. The injection volume was 10 m l. The retention times of the N dansyl Try Val Gly and Dansyl Try Val NH 2 were 1.3 and 2.35 minutes. Determination of initial rates of N dansyl Try Val NH 2 production as fixed N dansyl Try Val Gly concentration as a function of inhibitor concentration (Dixon analysis) Reactions at 37 C we re initiated by the addition of PAM (0.05 0.09 g) into 500 l of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v) ethanol, 0.001 % (v/v) Triton X 100, 10 m g/ml bovine catalase, 1.0 m M Cu (NO 3 ) 2 5.0 mM sodium ascorbate, inhibitors (1.0 2000 M) and 8 m M N d ansyl Try Val Gly. Aliquots of 50 m l were taken at 5, 10, 15, 20, and 25 minutes. The reaction aliquot was quenched with 10 m l of 6 % (v/v) trifluoroacetic acid in a HPLC microvial to terminate the PAM reaction. The aliquots were assayed for N dansyl Tr y Val Gly and dansyl Try Val NH 2 on a reverse phase C 18 column (100 mm x 4.6 mm, 5 m m particles, 120 pore). The mobile phase for the separation consisted of 100 mM sodium acetate, 55% deionized water and 45% acetonitrile.

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16 Determination of K M and V MAX values for N dansyl Tyr Val Gly as a function of inhibitor concentration Reactions at 37 C we re initiated by the addition of PAM (0.02 0.03 m g) into 500 l of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v) ethanol, 0.001 % (v/v) Triton X 100, 10 m g/ml bovine catalase, 1.0 m M Cu (NO 3 ) 2 5.0 mM sodium ascorbate, N dansyl Try Val Gly (1.25 12.5 m M), and inhibitor (20 800 m M). Aliquots of 50 m l were taken at 5, 10, 15, 20, and 25 minutes. The reaction aliquot was quenched with 10 m l of 6 % (v/v) trifluoroacetic acid in a HPLC microvial to terminate the PAM reaction. The aliquots were assayed for N d ansyl Try Val Gly and N dansyl Try Val NH 2 on a reverse phase C 18 column (100 mm x 4.6 mm, 5 m m particles, 120 pore). The mobile phase for the separation consisted of 100 mM sodium acetate, 55% deionized water and 45% acetonitrile. Determination of initial rates by oxygen consumption In order to determine kinetic constants (K M and V MAX ), rea ctions at 37 C were initiated by the addition of PAM (0.03 0.06 m g) into 2700 l of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v) ethanol, 0.001 % (v/v) Triton X 100, 10 m g/ml bovine catalase, 1.0 m M Cu (NO 3 ) 2 5.0 mM sodium ascorbate, and substrate (3.0 2500 M). V MAX values were normalized to controls ran at 11 mM N acetylglycine. Rates were monitored, recorded, and analyzed in Microsoft Excel. In order to determine the K i values (Dixon analysis), reactions at 37 C were initiated by the addition of PAM (0.03 0.06 m g) into 2700 l of 100 mM MES/NaOH pH

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17 6.0, 30 mM NaCl, 1.0% (v/v) ethanol, 0.001 % (v/v) Triton X 100, 10 m g/ml bovine catalase, 1.0 m M Cu (NO 3 ) 2 5.0 mM sodium ascorbate, 9 mM N acetylglycine, and inhibitor (1.0 200 M). Rates were monito red, recorded, and alayzed in Microsoft Excel. Copper Chelating Experiment Reactions at 37 C were initiated by the addition of 0.05 m g PAM into 500 l of 100 mM MES/NaOH pH 6.0, 30 mM NaCl, 1.0% (v/v) ethanol, 0.001 % (v/v) Triton X 100, 10 m g/ml bov ine catalase, 1.0 m M Cu (NO 3 ) 2, and 5.0 mM sodium ascorbate. Three dansyl Tyr Val Gly concentrations of 4 m M, 10 m M, and 20 m M were tested. S (4 methylthiobenzoyl)thioglycolic acid was tested at 12 m M and EDTA was tested at 30 m M. Both compounds were teste d at the three different N dansyl Tyr Val Gly concentrations. Aliquots of 50 m l were taken at 5, 10, 15, 20, and 25 minutes. The reaction aliquot was quenched with 10 m l of 6 % (v/v) trifluoroacetic acid in a HPLC microvial to terminate the PAM reaction. The aliquots were assayed for N dansyl Try Val Gly and N dansyl Try Val NH 2 on a reverse phase C 18 column (100 mm x 4.6 mm, 5 m m particles, 120 pore). The mobile phase for the separation consisted of 100 mM sodium acetate, 55% deionized water and 45% acetonitrile. Molecular Modeling HyperChem 7.5 was used for performing molecular modeling. Substrates and inhibitors were calculated to their lowest energy states using Amber 99. The compounds were then inserted into the active site of PHM in both the phenyl ring in (substrate like)

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18 and the phenyl ring out confirmations. The lowest energy conformation was determined by the method of Polak Ribiere. The energies were compared to the energy of the unbound reduced enzyme to determine the stability of the enzyme inhibitor complex. RESULTS AND DISCUSSION PAM substrates and inhibitors Results from this study (Table 2) suggest that compounds with sulfanyl type groups, such as S (phenylthioacetyl)thioglycolic acid and S (N phenylthiocarbamoyl) 3 mercaptopro pionic acid appear to bind more tightly then compounds with hydrogen or oxygen. (Phenylthio)acetic acid does not have a sulfanyl group and has a much higher K i Substrates with oxygen have a much higher K M then those with sulfanyls. This trend can be seen when comparing the K M of N (phenylthiopropionyl)glycine to N (6 phenylhexanoyl)glycine (Table 3). Since sulfur has a higher electron density than oxygen, it is possible that this atom interacts with the two coppers in the active site. In addition, s ince redox chemistry is necessary for the reaction to proceed and altering the coordinating sphere of copper ions could dramatically affect the reduction potentials, the sulfur atoms could interfere with electron transfer within the enzyme. Another class of compounds that exhibit tight binding contains other functional groups on the benzene ring. For example, methyl groups in the para position exhibit tight binding. S (4 Methylthiobenzoyl)thioglycolic acid has this moiety and has the low K i of 3.5 M. 4 Ethylhippuric acid and 4 propionylhippuric acid are two substrates with

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19 Inhibitor K i,app, ( m M) Inhibitor K i,app, ( m M) S S C O O H S (Thiobenzoyl)thioglycolic acid 39 5.2 S N H C O O H N (Phenylthioace tyl)alanine 3.6 0.4 S S C O O H S (4 Methylthiobenzoyl)thioglycolic acid 3.5 0.39 S S N C O O H 4 Cyano 4 methyl 4 thiobenzoyl sulfanyl butyric acid 20 5.8 S S C O C H 2 C H 3 S (4 Methylthiobenzoyl)thioglycolic acid ethyl ester 110 12 N H S S C O O H 2 S (N Phenylthiocarbamoyl) 3 mercaptopropionic acid 3.3 0.88 S S C O O H S (Phenylthioacetyl)thioglycolic acid 7.9 2.8 N H S S C O O H S (N Phenylthiocarbamoyl)thioglycolic acid 11 1.9 S S C O O H 2 S (3 Phenylthiopropionyl)thioglycolic acid 9.4 0.8 N H O O C O O H N Glycolic Acid phenyl urethane 54 4.0 O N H C O O H H S (D,L) Thiorphan 22 2.6 S C O O H (Phenylthio)acetic acid 380 36 S N O 2 C O O H (2 Nitrophenylthio)acetic acid 29 3.4 H 3 C C S C O O H S 1 0 S (Thiolauroyl)thioglycolate 0.54 0 .045 Table 2 Summary of inhibitors examined and corresponding inhibition constants. Inhibition constants were calculated by Dixon analysis.

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20 Substrate K M,app ( m M) V MAX,app ( m moles/min /mg) (V/K) app (M 1 s 1 ) Relative (V/K) app N H O C O O H Hippuric acid 1300 36 6.5 .063 6.2 10 3 1.0 N H O N H C O O H N Phenylhydantoic acid 350 70 7.6 0.65 2.7 10 4 4.4 N H O C O O H 4 Ethylhippuric acid 1162.1 166.45 2.9 0.2 2.8 10 3 0.45 S N H C O O H 2 N (Phenylthiopropionyl)glycine 38 15 1.3 0.11 4.3 10 4 6.9 N H O C O O H 2 4 Propionylhippuric Acid 1207.3 157.64 4.1 0.3 4.1 10 3 0.66 O N H C O O H 5 N (6 Phenylhexanoyl )glycine 96 14 8.2 0.53 1.1 10 5 17 O N H C O O H 7 N (8 Phenyloctanoyl)glycine 104.84 9.3273 6.4 0.2 1.2 10 5 19 Table 3 Summary of substrates examined and corresponding kinetic constants Kinetic constants were calcula ted using Michaelis Menton calculations.

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21 extended hydrocarbon chains in the para position. Interestingly this did not seem to affect the binding affinity. In addition, functional groups at the ortho position dramatically increase the binding affinity. ( 2 Nitrophenylthio)acetic acid has a 12 fold tighter binding affinity to (phenylthio)acetic acid. The length of the hydrocarbon chain also affects the binding affinity of the inhibitors. S (Thiobenzoyl)thioglycolic acid, S (phenylthioacetyl)thioglycolic a cid, and S (3 phenylthiopropionyl)thioglycolic acid have the same structure with the exception of the hydrocarbon chain before the sulfanyl group. The trend in binding affinity shows that in this group of compounds, the ideal length for inhibition is two carbon groups prior to the sulfanyl. The substrates N (6 phenylhexanoyl)glycine and N (8 phenyloctanoyl)glycine show that PAM prefers a lengthy hydrocarbon chain since their K M are 13 fold lower then hippurate. Figure 7 Different energies that can be p redicted by molecular modeling.

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22 Molecular modeling was performed to understand the relationship between K i and binding energies of the compounds to better understand the structure activity of the PAM. There are three types of energies to consider when det ermining the total free energy of a a .) G = G docking + G channel + G pocket b .) G pocket = H pocket T S pocket Figure 8 a.) The formula to calculate the total free energy for the complete docking of a compound into the active site of PHM. b.) T he formula for calculating the free energy for the pocket energy. Hyper Chem 7.5 calculates the pocket enthalpy. compound in the active site of PHM (Figure 7). The first of these energies is the docking energy. This is the energy for the compound to en ter the active site. The second is the channel energy, which is the energy of the compound to move through the active site channel to get to the binding site. The last is the pocket energy once inside the binding pocket (Figure 8a). The program used in this study, Hyperchem 7.5, is capable of calculating the channel enthalpies and the pocket enthalpies, which can be used to calculate the channel and pocket energies (Figure 8b). A more sophisticated software program is required to calculate the docking e nthalpies and actually proposing a specific path that the compound travels from entering the channel to binding into the pocket of the enzyme. For the preliminary studies only the pocket enthalpies were calculated for known substrates and inhibitors of PA M. The results from the molecular modeling can be seen in Table 4. The enthalpy of the reduced form of PHM without a substrate or inhibitor in the binding pocket was calculated. The enthalpies of the substrates and inhibitors were subtracted from the unb ound enthalpy to give the pocket enthalpy. The stability of a compound in the

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23 Inhibitor Bond Energy (kJ/mol) Inhibitor Bond Energy (kJ/mol) S S C O O H S (Thiobenzoyl)thioglycolic acid 10.88 S N H C O O H N (Phenylthioacetyl)alanine 16.91 S S C O O H *S (4 Methylthiobenzoyl)thioglycolic acid 20.92 S S N C O O H *4 Cyano 4 methyl 4 thiobenzoyl sulfanyl butyric acid 18.87 S S C O C H 2 C H 3 S (4 *Methylthiobenzoyl)thioglycolic acid ethyl ester 17.91 N H S S C O O H 2 *S (N Phenylthiocarbamoyl) 3 mercaptopropionic acid 16.51 S S C O O H S (Phenylthioacetyl)thioglycolic acid 15.11 N H S S C O O H *S (N Phenylthiocarbamoyl)thioglycolic acid 15.13 S S C O O H 2 S (3 Phenylthiopropionyl)thioglycolic acid 21.51 N H O O C O O H *N Glycolic Acid phenyl urethane 10.81 O N H C O O H H S (D,L) Thiorphan 38.37 S C O O H *(Phenylthio)acetic acid 9.99 S N O 2 C O O H (2 Nitrophenylthio)acetic acid 11.5 H 3 C C S C O O H S 1 0 S (Thiolauroyl)thioglycolate 19.07 Table 4 Summary of pocket enthalpies. The represents compounds that bind substrate like.

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24 binding site increases as calculated pocket enthalpy decreases. The substrates and inhibitors were calculated inside the binding site of PHM in two conformations. Th e phenyl group was placed going into the pocket, which is substrate like, and placed coming out of the pocket. The inhibitors showed stability in both conformations, Inhibitor Bond Energy (kJ/mol) Substrate Bond Energy (kJ/mol) S S C O O H S (Thiobenzoyl)thioglycolic acid 10.88 (39) N H O C O O H Hippuric acid 11.00 (1300) S S C O O H S (4 Methylthiobenzoyl)thioglycolic acid 20.92 (3.5) N H O C O O H 4 Methylhip puric acid 13.27 (1800) S S C O O H S (Phenylthioacetyl)thioglycolic acid 15.11 (7.9) O N H C O O H N (Phenylacetyl)glycine 14.95 (150) S S C O O H S (Phenylthioacetyl)thioglyc olic acid 15.11 (7.9) S N H C O O H N (Phenylthioacetyl)glycine 37.30 (24) S S C O O H 2 S (3 Phenylthiopropionyl)thioglycolic acid 21.51 (9.4) O N H C O O H 2 N (Hydrocinnamoyl)glycine 11. 96 (920) N H O O C O O H N Glycolic Acid phenyl urethane 10.81 (54) N H O N H C O O H N Phenylhydantoic acid 12.14 (350) Table 5 Comparison of inhibitor and substrate pocket enthalpies and binding constants. The nu mber in ( ) represents either K i for inhibitors or K M for substrates in M.

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25 depending on what substituents were on the phenyl ring. All of the substrates showed the preference of the phenyl ring going into the binding pocket. There is a correlation between pocket enthalpies and the binding constants. As a general trend, if the K i of an inhibitor is less then 10 M, the enthalpy predicted will be < 15. The active site contains two copper atoms, Cu H and Cu M which cycle through Cu II and Cu I redox s tates during catalysis. The two coppers are non equivalent, Cu M being the oxygen binding site and Cu H an electron donor site. All of the compounds gravitated toward Cu M This would suggest that there is an important interaction going on with this copper site. The enthalpies of some substrates and their corresponding inhibitors can be seen in Table 5. The pocket enthalpies of the substrates seem to coincide with their K M s, with the exception of 4 methyl hippuric acid. It shows more stability then hip puric acid inside the binding site even though it has a higher K M This does follow the trend of the inhibitors in which the hippuric acid derivative is a much worse inhibitor then the 4 methyl hippuric acid derivative. When the carbonyl is changed to a sulfanyl the compound becomes more stable in the binding pocket. There are a few compounds where the pocket enthalpies and the binding constants do not seem to correlate. (Phenylthioacetic) acid has a high binding constant, but its pocket enthalpy doe s not seem to be that much less then other compounds with lower binding constants. This compound may have a low docking enthalpy, so it can get into the channel, but may not bind to the substrate site well. This would effectively block substrates from bi nding to the enzyme. Based on the pocket enthalpy from 4 cyano 4 methyl 4 thiobenzoyl sulfanyl butyric acid, it would be expected that it would be a tight

PAGE 33

26 binding inhibitor, but it does not appear to be. This compound may not bind well to the active site (high docking enthalpy), but if it were able to access the substrate site, the enzyme inhibitor complex would have a relatively low K dissociation The same thing could be happening with thiorphan. Another possible reason for the differences in pocket en thalpies and binding constants may be that the compounds are binding to a different site on the enzyme other then the active site. In any case this shows that there is a high probability that there is more then one way a compound binds into the active s ite of PHM. When the substrates were tested using the dansyl assay and the oxygen consumption assay, the binding constants were found to be relatively close except for two compounds. 4 Ethylhippuric acid and 4 propionylhippuric acid have roughly a 4 fol d difference (Table 4) in K M This may be due to an unforeseen PAL inhibition or possible substrate inhibition. This may also indicate that the K M determined by oxygen Substrate K I,app Dansyl Assay ( m M) K M,app Oxygen consumption ( m M) N H O C O O H 4 Ethylhippuric Acid 370 37 1200 170 N H O C O O H 2 4 Propionylhippuric Acid 350 38 1200 160 Table 6 This is a comparison of K M value from the oxygen co nsumption assay and the K i value from the dansyl assay that show significant differences.

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27 consumption is greater then the true K d Copper chelating experiment An experiment was performed to determine if the sulfur containing inhibitors inhibit PAM by com peting with substrate for the active site or if they simply depleted copper from the enzyme through chelation. If the inhibitors were depleting the enzyme of copper, the resulting apo enzyme would be catalytically inactive and could not generate product f rom substrate at any concentration. On the other hand, if the compounds inhibited the enzyme, increasing the substrate concentration would out compete the inhibitor and product formation would increase as the as the substrate concentration increased. As seen in Graph 4, the inhibitor, S (4 methylthiobenzoyl)thioglycolic acid, showed an increase of product formation, as the substrate concentration was increased. To ensure that this result was accurate, the amount of inhibitor used was 3 times the K i Ther e was no product formation when EDTA was added to the reaction. Determination of K M and V MAX values for N dansyl Tyr Val Gly as a function of inhibitor concentration The results from a full inhibition study of S (3 phenyl thiopropionyl) thioglycolic ac id (Graph 5) indicated mixed inhibition, which was contradictory to previous models. The K is calculated was 25 10 M and the K ii was calculated to be 0.31 0.18 M. The

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28 Control-4uM Control-10uM Control-20uM EDTA -4uM EDTA -10uM EDTA -20uM Inhibitor-4uM Inhibitor-10uM Inhibitor-20uM Amount of Dansyl Tyr-Val-NH 2 (nmoles) 0 50 100 150 200 250 Graph 4 Plot of product formation vs. substrate concentration in 4, 10, and 20 M of EDTA and S (4 methylthiobenzoyl)thioglycolic acid (K i = 3.5 M). K is found in this experiment was similar to the K i calculated in previous experiments. The theory was that the inhibitors were competitive. To verif y the finding of mixed type inhibition, a known competitive substrate was tested in the dansyl assay. The results from the experiments with N octanoyl glycine (Graph 6) showed competitive inhibition with the N dansyl Tyr Val Gly with a K is of 550 64 M, which is similar to the K M measured by O 2 consumption (200 10 M ).

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29 -10 0 10 20 30 40 -1 -0.5 0 0.5 1 1/[S] (1/ M) 1/V (1/ mol/min/mg) no inhibitor 20 M 40 M 60 M S S C O O H Graph 5 Full kinetic study of S (3 phenyl thiopropionyl) thioglycolic acid. Conclusion The kinetic constants of several substrates and inhibitors of PAM were determined. Several inhibitors were shown to have inhibition constants in the low micromolar range and one, S (thiolauroyl)thioglycolate exhibited a submicromolar inhibition constant of 0.54 0.0045 M. Through molecular modeling, a preliminary tren d in the K i values was found. Inhibitors with a K i less then 10 M show a pocket enthalpy of less then 15.0 kJ/mol. Proposed molecules can be placed in the binding site of PHM to predict the K i The modeling also shows evidence of more then one binding mode. Some of the inhibitors follow the binding pattern of substrate, and other prefer to be oriented in the reverse position. The full kinetic study of S (3 phenyl thiopropionyl)

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30 thioglycolic acid supports the idea that some of these inhibitors exhibit more then one 1/[Substrate] (1/M) -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 1/Rate (1/mol/min/mg) 1 2 3 4 5 I = 0 M I = 200 M I = 400 M I = 600 M N H C O O H O Graph 6Full kinetic study of N-octanoyl glycine binding mode to PAM. S (3 phenyl thiopropionyl)thioglycolic acid is one of the inhibitors that shows a preference for the reverse conformation. Further modeling should be performed on additional compou nds to learn more about the structure activity relationship between binding compounds and PHM. Determination of channel enthalpies and docking enthalpies will also help show the pathway of binding. The inhibitors with the lowest inhibition constant were tested for cancer growth inhibition.

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31 Chapter Three Prostate Cancer Testing INTRODUCTION DU 145 Anti Proliferation Assays PAM is suspected to play an important role in androgen independent prostate cancer proliferation. Two androgen independent prostate cancer cell lines often used in cancer research are the DU 145 and PC 3 cells. The experiments discussed in this chapter utilize DU 145 androgen independent cells to probe the effects of PAM inhibitors on cell growth. Trypan blue stains only dead cells and thus, is used to assay for cell viability. This assay finds wide utilization in many types of cancer research (Lee2001). Another assay used to assay cell viability is dependent upon the production of NADH, a metabolic product of living cel ls. NADH production drives the reduction of a tetrazolium (MTT, being frequently employed) to an intensely colored formazan (Rocchi 2001). S (4 Methylthiobenzoyl)thioglycolic acid and S (phenylthioacetyl)thioglycolic acid were chosen for this study based on low inhibition constants and stability. S ( N Phenylthiocarbamoyl)thioglycolic acid and S ( N phenylthiocarbamoyl) 3 mercaptopropionic acid exhibit lower inhibtion constants, but cyclize at pH values < 10.

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32 S ( N Phenylthiocarbamoyl)thioglycolic acid has a t 1/2 of 30 minutes and S ( N phenylthiocarbamoyl) 3 mercaptopropionic acid has a t 1/2 of 8 hours at pH less than 10. N (4 Hydroxyphenyl) retinamide (4 HPR) was used as a control. This is a known growth inhibitor of DU 145 cells. The aminophenol ring and the long alkyl chain are two important structures on this compound that are key for drug cell contact (Takahashi 2002). The target enzyme that 4 HPR inhibits is not known. EXPERIMENTAL PROCEDURES Materials 0.05% Trypsin/ 0.53 mM EDTA w/o Ca, Mg, and NaHCO 3 minimum essential medium Eagle (EMEM), and phosphate buffered saline (PBS) were from Cellgro. Trypan blue (0.4% units) was from Life Technologies. Fetal bovine serum (FBS) was obtained from Atlanta Biologicals. Hanks Balanced Salt (HBS) was ob tained from ICN Biomedicals. DU 145 prostate cancer cell line was obtained from ATCC. Sodium pyruvate and sodium bicarbonate were obtained from Fisher. Cell culture was carried out in a Nuaire flow hood. Samples were observed under a Nilon TMS phase co ntrast microscope. Cells and cell culture The DU 145 prostate cancer cells were grown in Eagles minimum essential medium (EMEM) with Earles Salts (CaCl 2 KCl, MgSO 4 and Na 2 HPO 4 ) and 2 mM glutamine. The growth media also contained, 1.0 mM sodium py ruvate, 0.1 mM

PAGE 40

33 nonessential amino acids (contains all 20 amino acids), 1.5 g /L sodium bicarbonate, 10% FBS, 10,000 U/ml penicillin, and 10,000 g/ml of streptomycin (Papsidero 1981). Anti proliferation assay of S (4 methylthiobenzoyl)thioglycolic acid DU 145 cells were grown to 80% confluency in media as described. The cells we re then trypsonized, counted, and diluted to give 50,000 cells per well. The cells were allowed to attach to the bottom of the wells for 24 hours. The media was replaced and S (4 methylthiobenzoyl)thioglycolic acid was added to the media in concentration s of 0.1 mM, 0.5 mM, and 1 mM. S (4 methylthiobenzoyl)thioglycolic ethyl ester was tested at a concentration of 0.1 mM as a comparison. Cells were then counted at 24, 48, 72, and 96 hours. For each time point, the media was removed the cells and the cel ls were 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 (Lee 2001). Anti proliferation assay of S (phenylthioacetyl)thioglycolic acid DU 145 cells were grown to 80% confluency as described. Cells were trypsonized, counted, and di luted to give 50,000 cells per well. The cells were allowed to attach to the bottom of the wells for 24 hours. The media was replaced and S (phenylthioacetyl)thioglycolic acid was added to the media in concentrations of 0.5 mM, 1 mM, and 2 mM. 4 HPR was tested at a concentration of 0.01 mM as a positive control. Cells were then counted at 8, 24, 48, 72, and 96 hours. For each time point, the media

PAGE 41

34 was removed the cells and the cells were washed twice with PBS. Trypsin/EDTA was added to detached 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 appear ed blue (Lee 2001). RESULTS AND DISCUSSION Anti proliferation assay of S (4 methylthiobenzoyl)thioglycolic acid The results of the anti proliferative assay with S (4 methylthiobenzoyl) thioglycolic acid can be seen in Graph 7. S (4 Methylthiobenzoyl)th ioglycolic acid does inhibit the growth of androgen independent prostate cancer, but only at relatively high concentrations ( .0mM). There is only a significant decrease of growth at a concentration of 1 mM for S (4 methylthiobenzoyl)thioglycolic acid. This concentration is likely to be too high to be of any practical use as an anti cancer drug. To counter this problem, the ethyl ester of S (4 methylthiobenzoyl)thioglycolic acid was tested. It was thought that the ethyl ester would make the compound mo re hydrophobic and would facilitate transport across the cell membrane into the cell. Once inside the cell, an esterase would hydrolyze off the ethyl ester leaving S (4 methylthiobenzoyl) thioglycolic acid. As the results show, the ethyl ester was no mor e effective then the free acid as an anti proliferative agent. Since the K i of the ethyl ester is ~30 fold higher, these data suggest that the ethyl ester is more effective in crossing the cell membrane since the relative potency as an anti proliferative is approximately as the same as the free acid.

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35 No Inhibitor 0.1 mM 0.5 mM 1 mM Ethyl Ester at 0.1 mM % viable cells (% control) 0 20 40 60 80 100 120 24 hours 48 hours 72 hours 96 hours Graph 7 Anti proliferative assay of 4 methylthiobenzoylthioglycolic acid on DU 145 prostate cancer cells. The ethyl ester of this compound was also tested for comparison Cells were grown to 50,000 cells per well and then treated or not treated with var ious concentrations of 4 methylthiobenzoyl thioglycolic acid or its ethyl ester. Growth was measured at 24, 48, 72, and 96 hours. S S C O O H Anti proliferation assay of S (Phenylthioacetyl)thioglycolic acid Graph 8 shows the results of the anti proliferative assay with S (phenylthioacetyl)thioglycolic acid. There is a significant inhibition of growth with this compound at concentrations 1.0 mM. Again, the concentrations exhibiting anti proliferative activity preclude its use as an anti proliferative in humans. In this experiment, a known growth inhibitor of DU 145 cells was tested as a

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36 No Inhibitor 0.5 mM 1 mM 2 mM 4-HPR at 0.01 mM % viable cells (% control) 0 20 40 60 80 100 120 8 hours 24 hours 48 hours 72 hours 96 hours Graph 8 Anti proliferative assay of S (phenylthioacetyl)thioglycolic acid on DU 145 prostate cancer cells. 4 HPR was tested as a positive control. Cells were grown to 50,000 cells per well and then treated or not treated with various concentrations of S (phenylthioacetyl)thioglycolic acid or 4 HPR. Growth was measured at 8, 24, 48, 72, and 96 hours. S S C O O H positive control. 4 HPR completely inhibits cell growth in 48 hours at a concentration of 10 M, consistent with previously published results (Graph 8). S (4 Methylthiobenzoyl)thiogly colic acid and S (phenylthioacetyl)thioglycolic acid were also tested since they showed the lowest inhibition constant against PAM. Since 4 HPR completely stopped the growth of the DU 145 cells and S (4 methylthiobenzoyl) thioglycolic acid and S (phenylth ioacetyl)thioglycolic acid did not, it does not seem necessary to test the less potent inhibitors.

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37 Conclusion The anti proliferative assays on S (4 methylthiobenzoyl)thioglycolic acid and S (phenylthioacetyl)thioglycolic acid did show a significant in hibition of growth, but only at high concentrations. The high concentration needed to effectively treat androgen independent prostate cancer would require massive dosages if used to treat patients. Perhaps a compound with a lower inhibition constant woul d be more effective against the cells. These data suggest that a PAM inhibitor could be useful as an anti proliferative drug, but compounds > 10 3 fold more potent must be developed for anti PAM drugs to even be considered for clinical use. The kinetic con stants are known for many substrates and inhibitors of PAM. Though none of the compounds bind tight enough to PAM to completely stop the growth of androgen independent prostate cancer, they did show that they exhibit anti proliferative activity. This sho ws that there is a potential that PAM inhibitors of considerably higher potency (lower K i values) might have clinical relevance. Since only the S (thiolauroyl)thioglycolate bound at submicromolar concentrations, a new design should be developed. Instead of having substrate like inhibitors, product like or transition state analogs should be made. Preliminary data on some product like inhibitors have showed potential in attaining this goal. More extensive kinetic studies on all of the inhibitors are requ ired to fully understand the complexities of their interaction with PAM. Such data are necessary to completely exploit the specific interactions of this series of compounds with PAM to develop second generation compounds that bind more tightly to PAM

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38 Mor e intensive molecular modeling of the inhibitors into the active site should be done. With the kinetic constants of these compounds and others previously done, a better database can be constructed for future compound development. The database will help p redict which inhibitors will be most potent. The next step in the anti proliferative assays is to see if the compounds are actually inhibiting PAM. To do this, the amount of a particular amidated peptide hormone should be determined. If the compound is targeting PAM, there should be significantly less amount of the amidated hormone in the treated cancer cells compared to controls not exposed to the compound. The ethyl esters of current compounds need to be tested for esterase activity to see if they a re getting cleaved within the cell. If they are not, then perhaps another functional group can be attached to the compounds to facilitate the crossing of the cell membrane. For example a benzyl ester might yield more promising results.

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39 References B ell J; Ash D E; Snyder L M; Kulathila R; Blackburn N J; Merkler D J Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine alpha amidating enzyme BIOCHEMISTRY (1997), 36(51), 16239 46. Bertelsen A H; Beaud ry G A; Galella E A; Jones B N; Ray M L; Mehta N M Cloning and characterization of two alternatively spliced rat alpha amidating enzyme cDNAs from rat medullary thyroid carcinoma ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS (1990), 279(1), 87 96. Bonk hoff H; Stein U; Remberger K Androgen receptor status in endocrine paracrine cell types of the normal, hyperplastic, and neoplastic human prostate VIRCHOWS ARCHIV. A, PATHOLOGICAL ANATOMY AND HISTOPATHOLOGY (1993), 423(4), 291 4. Bradbury A F; Fin nie M D; Smyth D G Mechanism of C terminal amide formation by pituitary enzymes NATURE (1982), 298(5875), 686 8. Bundgaard H; Kahns A Chemical stability and plasma catalyzed dealkylation of peptidyl hydroxyglycine derivatives intermediates in peptide amidation. PEPTIDES (1991), 12, 745 748. Eipper B A; Mains R E; Glembotski C C Identification in pituitary tissue of a peptide alpha amidation activity that acts on glycine extended peptides and requires molecular oxygen, copper, and ascorb ic acid PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA (1983), 80(16), 5144 8. Eipper B A; Mains R E Recombinant DNA to study neuropeptide processing. General introduction ANNUAL REVIEW OF PHYSIOLOGY (1988), 50 305 7. Eipper B A; Stoffers D A; Mains R E The biosynthesis of neuropeptides: peptide alpha amidation ANNUAL REVIEW OF NEUROSCIENCE (1992), 15 57 85. Freeman J C; Villafranca J J; Merkler D J Redox cycling of enzyme bound copper during peptide amidation. JOURNAL OF THE AMERI CAN CHEMICAL SOCIETY (1993), 115(11), 4923 4.

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40 Jaron, S; Mains, R E; Eipper, B A; Blackburn, N J The Catalytic Role of the Copper Ligand H172 of Peptidylglycine a a Hydroxylating Monooxygenase (PHM): A Spectroscopic Stu dy of the H172A Mutant. BIOCHEMISTRY (2002), 41(44), 13274 13282. Jones B N; Tamburini P P; Consalvo A P; Young S D; Lovato S J; Gilligan J P; Jeng A Y; Wennogle L P A fluorometric assay for peptidyl alpha amidation activity using high performance liquid chromatography ANALYTICAL BIOCHEMISTRY (1988), 168(2), 272 9. Kyprianou N; English H F; Isaacs J T Programmed cell death during regression of PC 82 human prostate cancer following androgen ablation CANCER RESEARCH (1990), 50(12), 374 8 53. Landymore Lim A E; Bradbury A F; Smyth D G The amidating enzyme in pituitary will accept a peptide with C terminal D alanine as substrate BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (1983), 117(1), 289 93. Lee, H Protein kinase C involvement in aloe emodin and emodin induced apoptosis in lung carcinoma cell. BRITISH JOURNAL OF P HARMACOLOGY (2001), 134(5), 1093 1103. Matthews D E; Piparo K E; Burkett V H; Pray C C Animal Cell Technology: Products of Today, Prospects for To morrow (Spier, R.E., Griffiths, J.B. and Bethold, W., Eds.) Butterworth Heinemann Ltd., Oxford, U.K. (1994), 315 319. Merkler D J; Kulathila R; Tamburini P P; Young S D Selective inactivation of the hydroxylase activity of bifunctional rat peptidylglyci ne alpha amidating enzyme ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS (1992), 294(2), 594 602. Merkler, D J; C terminal amidated peptides: production by the in vitro enzymic amidation of glycine extended peptides and the importance of the amide to bi oactivity. ENZYME AND MICROBIAL TECHNOLOGY (1994), 16(6), 450 6. Merkler D J; Kulathila R; Francisco W A; Ash D E; Bell J The irreversible inactivation of two copper dependent monooxygenases by sulfite: peptidylglycine a a amidating enzyme and dopami ne b b monooxygenase. FEBS LETTERS (1995), 366(2,3), 165 9. Merkler K A; Baumgart L E; DeBlassio J L; Glufke U; King L 3rd; Ritenour Rodgers K; Vederas J C; Wilcox B J; Merkler D J A pathway for the biosynthesis of fatty acid amides ADVANCES IN E XPERIMENTAL MEDICINE AND BIOLOGY (1999), 469 519 25.

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41 Miller D A; Sayad K U; Kulathila R; Beaudry G A; Merkler D J; Bertelsen A H Characterization of a bifunctional peptidylglycine alpha amidating enzyme expressed in Chinese hamster ovary cells ARC HIVES OF BIOCHEMISTRY AND BIOPHYSICS (1992), 298(2), 380 8. Papsidero L D; Kuriyama M; Wang M C; Horoszewicz J; Leong S S; Valenzuela L; Murphy G P; Chu T M Prostate antigen: a marker for human prostate epithelial cells JOURNAL OF THE NATIONAL CAN CER INSTITUTE (1981), 66(1), 37 42. Parker S L; Tong T; Bolden S; Wingo P A Cancer statistics, 1996 CA: A CANCER JOURNAL FOR CLINICIANS (1996), 46(1), 5 27. Rocchi P; Boudouresque F; Zamora A J; Muracciole X; Lechevallier E; Martin P; Ouafik, L'Houcine. Expression of adrenomedullin and peptide amidation activity in human prostate cancer and in human prostate cancer cell lines. CANCER RESEARCH (2001), 61(3), 1196 1206. Segel, I Biochemical Calculations: How to Solve Mathematical Prob lems in General Biochemistry. 2nd Ed. (1976), 448. Southan C; Kruse L I Sequence similarity between dopamine beta hydroxylase and peptide alpha amidating enzyme: evidence for a conserved catalytic domain FEBS LETTERS (1989), 255(1), 116 20. Spector, T; Cleland, W W Meanings of Ki for conventional and alternate substrate inhibitors. BIOCHEMICAL PHARMACO LOGY (1981), 30(1), 1 7. Takahashi N; Ohba T; Togashi S; Fukui T. Biological Activity of p Methylaminophenol, an Essential Structural Component of N (4 Hydroxyphenyl)retinamide, Fenretinide JOURNAL OF BIOCHEMISTRY (2002), 132(5), 767 74. Tamburini P P; Young S D; Jones B N; Palmesino R A; Consalvo A P Peptide substrate specificity of the alpha amidating enzyme isolated from rat medullary thyroid CA 77 cells INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH (1990), 35(2), 153 6. Wilcox B J; Ritenour Rodgers K J; Asser A S; Baumgart L E; Baumgart M A; Boger D L; DeBlassio J L; deLong M A; Glufke U; Henz M E; King L 3rd; Merkler K A; Patterson J E; Robleski J J; Vederas J C; Merkler D J N acylglycine amidation: implications for the biosynthesis of fatty acid primary amides BIOCHEMISTRY (1999), 38(11), 3235 45.


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Substrate-based inhibitors of peptidylglycine -amidating monooxygenase (PAM) as anti-proliferative drugs for cancer
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ABSTRACT: C-Terminal glycine-extended prohormones are enzymatically converted to -amidated peptides, by peptidylglycine -amidating monooxygenase (PAM). PAM is a bifunctional enzyme with two catalytic domains: peptidylglycine -hydroxylating monooxygenase (PHM) and peptidylglycine peptidylglycineaminoglycolate lyase (PAL). PAM has a significant role in the proliferation of androgen-independent prostate cancer. Thus, the inhibition of PAM could halt cancer growth. Hippurate and hippurate analogs were used as lead compounds for developing inhibitors for PAM. The hippurate analogs exhibiting the highest affinity to PAM (lowest inhibition constant) did inhibit the growth of human androgen-independent prostate cancer DU 145 cells.
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