Catalytically active peptidylglycine alpha-amidating monooxygenase in the media of androgen-independent prostate cancer cell lines.

Peptidylglycine alpha-amidating monooxygenase (PAM) converts inactive terminal-glycine prohormones into their activated alpha-amidated forms. PAM is thought to play a role in the development of antiandrogen drug resistance in prostate cancer (CaP) through PAMactivated autocrine growth. On the basis of the previous finding that many lung cancer cell lines excrete PAM into their culture media, this study investigates PAM levels in media collected from human CaP cell line cultures.

Microplate screening of the differential effects of test agents on Hoechst 33342, rhodamine 123, and rhodamine 6G accumulation in breast cancer cells that overexpress P-glycoprotein.

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level.

An investigation of position 3 in arginine vasopressin with aliphatic, aromatic, conformationally-restricted, polar and charged amino acids.

We report the solid-phase synthesis and some pharmacological properties of 23 new analogs of arginine vasopressin (AVP) which have the Phe3 residue replaced by a broad variety of amino acids. Peptides 1-9 have at position 3: (1) the mixed aromatic/aliphatic amino acid thienylalanine (Thi) and the aliphatic amino acids; (2) cyclohexylalanine (Cha); (3) norleucine (Nle); (4) Leu; (5) norvaline (Nva); (6) Val; (7) alpha-aminobutyric acid (Abu); (8) Ala; (9) Gly. Peptides 10-23 have at position 3: the aromatic amino acids, (10) homophenylalanine (Hphe): (11) Tyr; (12) Trp; (13) 2-naphthylalanine (2-Nal); the conformationally-restricted amino acids (14) Pro; (15) 2-aminotetraline-2-carboxylic acid (Atc); the polar amino acids (16) Ser; (17) Thr; (18) Gln; and the charged amino acids (19) Asp; (20) Glu; (21) Arg; (22) Lys; (23) Orn.

Ethosuximide is primarily metabolized by CYP3A when incubated with isolated rat liver microsomes.

The cytochrome P450 (CYP) subfamily responsible for ethosuximide metabolism was investigated by HPLC assay of ethosuximide incubations with isolated rat liver microsomes from control rats and from rats treated with inducing agents to enrich hepatic microsomes in selected CYP isoforms. Inducing agents included beta-naphthoflavone (BNF, CYP1A inducer), phenobarbital (PB, CYP2B/2C/3A), isoniazid (INH, CYP2E1), clotrimazole (CTZ, CYP3A), clofibrate (CLO, CYP4A), and an imidazole CTZ-analog known as CDD3543 (CYP3A). Incubations with BNF, INH, CTZ, and control microsomes showed significantly (p<0.

Position three in vasopressin antagonist tolerates conformationally restricted and aromatic amino acid substitutions: a striking contrast with vasopressin agonists.

We report the solid-phase synthesis and some pharmacological properties of 12 position three modified analogues (peptides 1-12) of the potent non-selective antagonist of the antidiuretic (V2-receptor), vasopressor (V1a-receptor) responses to arginine vasopressin (AVP) and of the uterine contracting (OT-receptor) responses to oxytocin (OT), [1(-beta mercapto-beta,beta-pentamethylenepropionic acid)-2-O-ethyl-D-tyrosine 4-valine] arginine vasopressin [d(CH2)5D-Tyr(Et)2VAVP] (A) and two analogues of (B) (peptides 13,14), the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid3 (Tic3) analogue of (A). Peptides 1-12 have the following substituents at position three in (A): (1) Pro; (2) Oic; (3) Atc; (4) D-Atc; (6) D-Phe; (7) Ile; (8) Leu; (9) Tyr; (10) Trp; (11) Hphe; (12) [HO]Tic; Peptide (13) is the Tyr-NH2(9) analogue of (B): Peptide (14) is the D-Cys(6) analogue of (B). All 14 new peptides were evaluated for agonistic and antagonistic activities in in vivo V2 and V1a assays and in vitro (no Mg2+)n oxytocic assays.

Effects of a D-Cys6/L-Cys6 interchange in nonselective and selective vasopressin and oxytocin antagonists.

We report the solid-phase synthesis of the D-Cys6 analogues of arginine-vasopressin (AVP), peptide 1, of the selective AVP vasopressor (V1a receptor) antagonist [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-O-methyltyrosine]arginine-vasopressin (d(CH2)5[Tyr(Me)2]-AVP, (A)), peptide 2, of the three nonselective antidiuretic/vasopressor (V2/V1a receptor) AVP antagonists d(CH2)5[Tyr(Et)2]VAVP (B), d(CH2)5[D-Tyr(Et)2]VAVP (C), and d(CH2)5[D-Phe2]VAVP (D) (where V = Val4), peptides 3-5, of the nonselective oxytocin (OT) antagonists d(CH2)5-[Tyr(Me)2]OVT (E) and d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT (F) (where OVT = ornithine-vasotocin), peptides 6 and 7, and of the selective OT antagonists desGly-NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT (G) and d(CH2)5]D-Trp2,Thr4]OVT (H), peptides 8 and 9. We also present the repeat syntheses of the previously reported d(CH2)5[D-Trp2]AVT (peptide 10) and its D-Cys6 analogue (peptide 11) (where AVT = arginine-vasotocin). Peptides 1-11 were assayed for agonistic and antagonistic activities in in vivo V1a, V2, and oxytocic assays and in in vitro oxytocic assays without and with 0.

An exploration of the effects of L- and D-tetrahydroisoquinoline-3-carboxylic acid substitutions at positions 2, 3 and 7 in cyclic and linear antagonists of vasopressin and oxytocin and at position 3 in arginine vasopressin.

We have investigated the effects of mono-substitutions with the conformationally restricted amino acid, 1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid (Tic) at position 3 in arginine vasopressin (AVP), at positions 2, 3 and 7 in potent non-selective cyclic AVP V2/V1a antagonists, in potent and selective cyclic and linear AVP V1a antagonists, in a potent and selective oxytocin antagonist and in a new potent linear oxytocin antagonist Phaa-D-Tyr(Me)-Ile-Val-Asn-Orn-Pro-Orn-NH2 (10). We report here the solid-phase synthesis of peptide 10 together with the following Tic-substituted peptides: 1. [Tic3]AVP: 2.

Advances in the design of selective antagonists, potential tocolytics, and radioiodinated ligands for oxytocin receptors.

Despite intensive efforts over three decades in many laboratories, attempts to design peptide antagonists of oxytocin (OT) which are more selective for OT uterine receptors than for vasopressin (AVP), vasopressor V1a receptors, have met with only limited success. We will review the current status of the field and report on studies in our laboratories which have led to the design of highly potent non-selective and selective OT antagonists. Virtually all are more potent (2-6 fold) and a number are more selective (10-12 fold) than Atosiban, currently in clinical trial as a tocolytic agent.

Design of potent and selective linear antagonists of vasopressor (V1-receptor) responses to vasopressin.

We report the solid-phase synthesis of 21 linear analogues of A and D, two nonselective antagonists of the vasopressor (V1) and antidiuretic (V2) responses to arginine vasopressin (AVP). A is Aaa-D-Tyr(Et)-Phe-Val-Asn-Abu-Pro-Arg-Arg-NH2 (where Aaa = adamantylacetyl at position 1). D is the des-Arg9 analogue of A.

Design and synthesis of a peptide having chymotrypsin-like esterase activity.

A peptide having enzyme-like catalytic activity has been designed and synthesized. Computer modeling was used to design a bundle of four short parallel amphipathic helical peptides bearing the serine protease catalytic site residues serine, histidine, and aspartic acid at the amino end of the bundle in the same spatial arrangement as in chymotrypsin (ChTr). The necessary "oxyanion hole" and substrate binding pocket for acetyltyrosine ethyl ester, a classical ChTr substrate, were included in the design.

Solid-phase synthesis of 16 potent (selective and nonselective) in vivo antagonists of oxytocin.

We describe the synthesis and some pharmacological properties of 16 new in vivo antagonists of oxytocin. These are based on modifications of three peptides: A, B, and C. A is our previously reported potent and selective antagonist of the vasopressor (V1 receptor) responses to arginine-vasopressin (AVP)/weak oxytocin antagonist, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid), 2-O-methyltyrosine]arginine-vasopressin (d(CH2)5[Tyr(Me)2]AVP.

Novel linear antagonists of the antidiuretic (V2) and vasopressor (V1) responses to vasopressin.

We report the solid phase synthesis of a series of 16 linear analogues of the cyclic antagonist of the antidiuretic (V2) and the vasopressor (V1) responses to arginine vasopressin (AVP), d(CH2)5[D-Tyr(Et)2, Val4]AVP(A). Peptide 1, the linear precursor of (A), (CH2)5(SH)-CH2-CO-D-Tyr(Et)-Phe-Val-Asn-Cys-Pro-Arg-Gly-NH2 was modified at position six with alpha-L-aminobutyric acid (Abu) to give peptide 2. Further modifications of the Abu6 analogue (No.

Potent V2 vasopressin antagonists with structural changes at their C-terminals.

A variety of structural changes were made in the C-terminals of four potent antidiuretic (V2) antagonists. The parent analogs were all derivatives of [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)]arginine-vasopressin, d(CH2)5AVP, namely d(CH2)5[D-Phe2,Ile4]AVP, d(CH2)5[D-Ile2,Ile4]AVP, d(CH2)5[D-Tyr(Et)2, Val4]AVP and d(CH2)5[D-Tyr(Et)2,Ile4]AVP. A number of amino acid amides were substituted for the C-terminal 9-glycinamide without reducing their V2-antagonistic potencies in rats.

C-terminal deletions in agonistic and antagonistic analogues of vasopressin that improve their specificities for antidiuretic (V2) and vasopressor (V1) receptors.

We report the solid-phase synthesis of 12 desGly and 12 desGly(NH2) analogues of arginine-vasopressin (AVP), two highly selective antidiuretic (V2) agonists, four vasopressor (V1) antagonists, and five V2/V1 antagonists. The parent AVP agonists are (1) AVP, (2) 1-deamino[8-D-arginine]vasopressin (dDAVP), and (3) its 4-valine analogue, dVDAVP. The parent V1 antagonists are (4) [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid)] arginine-vasopressin (d(CH2)5AVP), (5) d(CH2)5VDAVP, (6) [1-deaminopenicillamine,4-valine,8-D-arginine]vasopressin (dPVDAVP), and (7) d(CH2)5[Tyr(Me)]AVP.

No requirements of cyclic conformation of antagonists in binding to vasopressin receptors.

Early reports that acyclic analogues of oxytocin and vasopressin (AVP) have drastically reduced agonistic activities established as dogma that an intact hexapeptide ring structure is essential for the pharmacological activities of analogues of neurohypophysial hormones. Thus, virtually all the many hundreds of agonistic and antagonistic analogues of the neurohypophysial peptides that have been reported contain an intact ring. Here we report that an intact ring is not essential for binding of antagonistic AVP analogues to vasopressor (V1) or antidiuretic (V2) AVP receptors.

Carboxy terminus of vasopressin required for activity but not binding.

Vasopressin antagonists are valuable pharmacological tools for investigating physiological and behavioural functions of the nonapeptide arginine-vasopressin (AVP). The removal of glycinamide from the carboxy terminus of AVP drastically reduces its characteristic vasopressor and antidiuretic activities. In contrast to this we show here that removal of the carboxy-terminal glycinamide or the glycine at position 9 from several vasopressin antagonists makes little difference to their ability to block vasopressor and antidiuretic responses to AVP.

Potent and selective antagonists of the antidiuretic responses to arginine-vasopressin based on modifications of [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-D-isoleucine,4- valine]arginine-vasopressin at position 4.

As part of a program in which we are attempting (a) to obtain more potent and/or more selective antagonists of the antidiuretic responses to arginine-vasopressin (AVP) and (b) to delineate the structural features at positions 1-9 required for antidiuretic antagonism, we have synthesized 13 new analogues of the antidiuretic antagonist [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-D-isoleucine,4- valine]arginine-vasopressin [d(CH2)5[D-Ile2]VAVP] in which the valine residue at position 4 has been replaced by the L-amino acids Abu, Ile, Thr, Ala, Ser, Nva, Gln, Leu, Lys, Cha, Asn, Orn, and Phe and two new analogues of the antidiuretic antagonist [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid),2-D-phenylalanine,4- valine]arginine-vasopressin [d(CH2)5[D-Phe2]VAVP] with the Val4 residue replaced by Ser and Orn. These analogues are 1, d(CH2)5[D-Ile2,Abu4]AVP; 2, d(CH2)5[D-Ile2,Ile4]AVP; 3, d(CH2)5[D-Ile2,Thr4]AVP; 4, d(CH2)5[D-Ile2,Ala4]AVP; 5, d(CH2)5[D-Ile2,Ser4]AVP; 6, d(CH2)5[D-Ile2,Nva4]AVP; 7, d(CH2)5[D-Ile2]AVP; 8, d(CH2)5[D-Ile2,Leu4]AVP; 9, d(CH2)5[D-Ile2,Lys4]AVP; 10, d(CH2)5[D-Ile2,Cha4]AVP; 11, d(CH2)5[D-Ile2,Asn4]AVP; 12, d(CH2)5[D-Ile2,Orn4]AVP; 13, d(CH2)5[D-Ile2,Phe4]AVP; 14, d(CH2)5[D-Phe2,Ser4]AVP; and 15, d(CH2)5[D-Phe2,Orn4]AVP. The protected peptide precursors for these peptides were prepared by the solid-phase method, followed by ammonolytic cleavage.

Potent antagonists of the antidiuretic responses to arginine-vasopressin based on modifications of [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-D- phenylalanine,4-valine]arginine-vasopressin at position 4.

As part of a program in which we are attempting (a) to delineate the structural features at positions 1-9 in our previously reported antidiuretic antagonists required for antidiuretic antagonism and (b) to obtain analogues with enhanced antiantidiuretic potency and/or selectivity, we have synthesized 14 new analogues of the antidiuretic antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-D-phenylalanine,4-valine]arginine-vasopressin [d-(CH2)5-D-Phe2VAVP), in which the valine residue at position 4 was replaced by the following L-amino acids and glycine: Ile, Abu, Thr, Ala, Gln, Lys, Cha, Nle, Nva, Phe, Leu, Gly, Tyr, and Pro. These analogues are 1, d-(CH2)5-D-Phe2,Ile4AVP; 2, d(CH2)5-D-Phe2,Abu4AVP; 3, d(CH2)5-D-Phe2,Thr4AVP; 4, d(CH2)5-D-Phe2,Ala4AVP;5, d(CH2)5-D-Phe2AVP; 6, d(CH2)5-D-Phe2,Lys4AVP; 7, d(CH2)5-D-Phe2,Cha4AVP; 8, d(CH2)5-D-Phe2,Nle4AVP; 9, d(CH2)5-D-Phe2,Nva4AVP; 10, d(CH2)5-D-Phe2,Phe4AVP; 11, d(CH2)5-D-Phe2,Leu4AVP; 12, d(CH2)5-D-Phe2,Gly4AVP; 13, d(CH2)5-D-Phe2,Tyr4AVP; 14, d(CH2)5-D-Phe2,Pro4AVP. The protected intermediates required for the synthesis of all of these peptides were prepared by the solid-phase method and cleaved from the resin by ammonolysis.

Design of more potent antagonists of the antidiuretic responses to arginine-vasopressin.

As part of a program aimed at designing more potent and selective antagonists of the antidiuretic responses to arginine-vasopressin (AVP), we substituted O-alkyl-D-tyrosine (where alkyl = methyl, ethyl, isopropyl, or n-propyl) at position 2 in our eight previously reported O-alkyl-L-tyrosine antagonists of antidiuretic and vasopressor responses to AVP. We also substituted D-tyrosine for L-tyrosine in two vasopressor antagonists with weak antidiuretic agonistic activity, [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),4-valine,8-D-arginine]vasopressin [d(CH2)5VDAVP] and its L-arginine isomer [d(CH2)5VAVP]. The ten analogues, synthesized by the solid-phase method, are as follows: 1, d(CH2)5-D-Tyr(Me)VDAVP; 2, d(CH2)5-D-Tyr(Et)VDAVP; 3, d(CH2)5-D-Tyr(i-Pr)VDAVP; 4, d(CH2)5-D-Tyr(n-Pr)VDAVP; 5, d(CH2)5-D-Tyr(Me)VAVP; 6, d(CH2)5-D-Tyr(Et)VAVP; 7, d(CH2)5-D-Tyr(n-Pr)VAVP; 8, d-(CH2)5-D-Tyr(i-PR)VAVP; 9, d(CH2)5-D-TyrVDAVP; 10, d(CH2)5-D-TyrVAVP.

Conformation of disulfide bridges in peptides with pepsin partial sequences.

On the basis of Raman spectra investigation of two model heterodetic cyclic peptides, containing partial sequences of pepsin fragments 45--50 and 206--210 of the chain, it was concluded that the disulfide bridge conformation in pepsin is determined not only by the size and conformation of the peptide loops created by disulfide bridges, but also by the peptide fragments located outside these loops.