Rodney L. Johnson

An efficient and high yield method for the N-tert-butoxycarbonyl protection of sterically hindered amino acids

Abstract An improved method for the N - tert -butoxycarbonyl protection of the amino functionality of α-alkylated prolines and other sterically hindered α,α-disubstituted amino acids has been developed in which the lipophilic base tetramethylammonium hydroxide is used to solubilize the otherwise insoluble zwitterionic amino acid in acetonitrile, thereby obviating the need for an aqueous medium.

Interaction of l‐Prolyl‐l‐Leucyl Glycinamide with Dopamine D2 Receptor: Evidence for Modulation of Agonist Affinity States in Bovine Striatal Membranes

The role of the hypothalamic tripeptide l‐prolyl‐l‐leucyl‐glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high‐affinity agonist, n‐propylnor‐apomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose‐dependent manner, the maximum effect being observed at 10‐‐7‐‐10‐‐6M concentration of the tripeptide. The Scat‐chard analysis of [3H]NPA binding to membranes preincu‐bated with 10‐‐6M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high‐ and low‐affinity states of the receptor displayed an increase in the population and affinity of the high‐affinity form of the receptor with PLG treatment. The low‐affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5′‐guanylyl‐imidodiphosphate [Gpp(NH)p]‐induced inhibition of high‐affinity agonist binding was also observed, as the ratio of high‐ to low‐affinity forms of the receptor was significantly higher in the PLG‐treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p‐induced inhibition of agonist binding by increasing the EC50 of the nu‐cleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high‐affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p. The binding of antagonists and agonists to α2 adrenergic receptors, negatively coupled to adenylate cyclase in the striatum. was not affected by PLG. The results suggest that PLG modulates the affinity states of the dopamine D2 receptor, possibly by enhancing its interaction with the guanine nucleotide regulatory protein.

Mesolimbic and striatal dopamine receptor supersensitivity: Prophylactic and reversal effects of L-prolyl-L-leucyl-glycinamide (PLG) ☆

Functional supersensitivity of mesolimbic and striatal dopamine receptors has been suggested to contribute to the pathogenesis of schizophrenia and tardive dyskinesia. Using the rodent model of chronic administration of the neuroleptic haloperidol, we investigated the possible desensitizing effects of a tripeptide structurally unrelated to dopamine agonists, L-prolyl-L-leucyl-glycinamide (PLG) on mesolimbic and striatal dopaminergic receptor supersensitivity. Administration of PLG either prior to or after chronic haloperidol, inhibited the supersensitivity of dopamine receptors. The results have implications for pharmacological intervention in preventing tardive dyskinesia and relapse psychosis of schizophrenia.

Structure - function relationships for gamma-substituted glutamate analogues on dentate granule cells.

We previously demonstrated in the Schaffer collateral-CA1 region of the hippocampus that bath-applied agonists could be distinguished from antagonists among a group of acidic amino acid analogues by extracellular recording techniques. Here we report the use of the extracellular signs of agonist activity for discerning agonists and antagonists among several gamma-substituted glutamate analogues tested in the perforant path. The two-pathway composition of the perforant path offers the advantage over CA1 in that pathway-specificity, a postulated characteristic of antagonists, may be tested. By extracellular recording, D- and L-homocysteic acid, L-serine-O-sulfate, and L-2-amino-4-(5-tetrazolyl)-butanoic acid [L-glutamate tetrazole] were identified as agonists, and all 4 analogues were more potent than L-glutamate for inhibiting synaptic field potentials. Two previously identified antagonists, L-2-amino-4-phosphonobutyric acid and L-O-phosphoserine, exhibited the pathway-specificity and inhibitory kinetics consistent with properties expected for antagonists; both compounds detected 3 perforant path components with the same rank in sensitivity, suggesting that they are acting on the same set of receptors.

MODULATION OF DOPAMINE RECEPTOR AGONIST-INDUCED ROTATIONAL BEHAVIOR IN 6-OHDA-LESIONED RATS BY A PEPTIDOMIMETIC ANALOGUE OF PRO-LEU-GLY-NH2 (PLG)

The present study was undertaken to determine if the previously reported in vitro interactions of the Pro-Leu-Gly-NH2 (PLG) peptidomimetic analogue 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacet amide (PAOPA) with the dopaminergic system could be exhibited in an in vivo animal model using 6-hydroxydopamine (6-OHDA)-lesioned rats. In this model, PAOPA was found to potentiate the contralateral rotational behavior induced by either apomorphine or L-DOPA. PAOPA was 100-fold more potent than PLG, and produced a fourfold greater response than PLG when administered i.p. PAOPA also potentiated contralateral rotations induced by SKF-38393 and quinpirole. In summary, the results of this study indicate that PAOPA, a conformationally constrained peptidomimetic analogue of PLG, can modulate dopaminergic activity in vivo with higher potency and efficacy than PLG.

Effects of quisqualic acid analogs on metabotropic glutamate receptors coupled to phosphoinositide hydrolysis in rat hippocampus

L-Glutamic acid (L-Glu) and L-aspartic acid (L-Asp) activate several receptor subtypes, including metabotropic Glu receptors coupled to phosphoinositide (PI) hydrolysis. Quisqualic acid (Quis) is the most potent agonist of these receptors. There is evidence that activation of these receptors may cause a long lasting sensitization of neurons to depolarization, a phenomenon called the Quis effect. The purpose of the current studies was to use Quis analogs and the recently identified metabotropic receptor antagonist, (+)-alpha-methyl-4-carboxy-phenylglycine((+)-MCPG), to define the structural properties required for interaction with the metabotropic receptors coupled to PI hydrolysis and to determine if the Quis effect is mediated by these receptors. The effects of Quis analogs on PI hydrolysis were studied in the absence or presence of the metabotropic receptor-specific agonist 1SR,3RS-1-amino-1,3-cyclopentanedicarboxylic acid (1SR,3RS-ACPD) in neonatal rat hippocampus. Some of the compounds that induce the Quis effect also stimulate PI hydrolysis, including Quis itself and 9 (homoquisqualic acid). Not all of the Quis analogs that stimulate PI hydrolysis, however, induce the Quis effect, including 7A (EC50 = 750 +/- 150 microM) and (RS)-4-bromohomoibotenic acid (BrHI) (EC50 = 130 +/- 40 microM). Although (+)-MCPG blocked PI hydrolysis stimulated by Quis (IC50 = 370 +/- 70 microM), it had no effect on the induction of the Quis effect. Other Quis analogs did not stimulate PI hydrolysis but rather blocked the effects of 1SR,3RS-ACPD. The IC50 values were 240 +/- 70 microM for 2, 250 +/- 90 microM for 3, and 640 +/- 200 microM for 4. Data for inhibition by 2 and 3 were consistent with non-competitive mechanisms of action. These studies provide new information about the structural features of Quis required for interaction with metabotropic receptors coupled to PI hydrolysis and provide evidence that the Quis effect is not mediated by (+)-MCPG sensitive subtypes of these receptors.

Modulation of high-affinity CNS dopamine D2 receptor by l-pro-l-leu-glycinamide (PLG) analogue 3(R)-(N-l-prolylamino)-2-oxo-1-pyrrolidineacetamide

1. In our previous studies, we reported that one of the conformationally constrained analogues of PLG, 3(R)-(N-L-prolylamino)-2-oxo-1-pyrrolidineacetamide (PAOPA), was found to be extremely potent in enhancing the [3H]ADTN binding when membranes were preincubated with this compound. The PAOPA was without effect when directly added to assay tubes. In this study, the compound PAOPA was examined on agonist binding to high affinity state of the dopamine D2 receptor. 2. The potent analogue PAOPA was able to prevent the GTP-induced conversion of high affinity state of dopamine D2 receptor to low affinity state. 3. PLG analogue PAOPA modulates the affinity states of the dopamine D2 receptor possibly by affecting its interaction with the G-protein(s).

Allosteric Modulation of the Dopamine D2 Receptor by Pro-Leu-Gly-NH2 Peptidomimetics Constrained in Either a Polyproline II Helix or a Type II β-Turn Conformation

Type II beta-turn mimics and polyproline II helix mimics based upon diastereoisomeric 5.6.5 spiro bicyclic scaffolds have provided two pairs of Pro-Leu-Gly-NH(2) (PLG) peptidomimetics with contrasting dopamine receptor modulating activities. Compounds 1a and 3a were found to be positive allosteric modulators of the dopamine receptor, while the corresponding diastereoisomeric compounds 1b and 3b provided the first PLG peptidomimetics with the ability to decrease the binding of agonists to the dopamine receptor. The positive allosteric modulating activity of 3a supported the hypothesis that a polyproline II helix conformation is the bioactive conformation for the PLG analogue Pro-Pro-Pro-NH(2). The results also show that a change in the bridgehead chirality of the 5.6.5 scaffold brings about opposite effects in terms of the modulation of the dopamine receptor.

Pro-Leu-glycinamide and its peptidomimetic, PAOPA, attenuate haloperidol induced vacuous chewing movements in rat: A model of human tardive dyskinesia.

In the present experimental paradigm, we examine the effect of L-prolyl-L-leucyl-glycinamide (PLG) co-administration with haloperidol on vacuous chewing movements (VCM) in rats-a model of tardive dyskinesia (TD) in humans. We examined the dose dependent induction of VCM through both injected and orally administered PLG (MIF-1). Our results show significant levels of VCM attenuation (P<0.05) in rats treated with 10mg/kg of PLG. Doses of 1 and 100mg/kg were ineffective. Reductions were present in both orally treated and injected rats. We also examined the therapeutic effect of a peptidomimetic of PLG-PAOPA. PAOPA was able to produce similar behavioral effects to PLG at a dose, which was 100-fold lower than the effective dose of PLG. These results suggest that PLG may play a role in D2 receptor expression and function, as well as providing a therapy for neuroleptic induced TD.

PAOPA, a potent analogue of Pro-Leu-glycinamide and allosteric modulator of the dopamine D2 receptor, prevents NMDA receptor antagonist (MK-801)-induced deficits in social interaction in the rat: Implications for the treatment of negative symptoms in schizophrenia

The aim of this study was to investigate whether a potent analogue of the endogenous brain peptide l-prolyl-l-leucyl-glycinamide (PLG), (3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA), can prevent the induction of social withdrawal caused by sub-chronic treatment with the non-competitive NMDA (N-methyl-l-aspartate) receptor antagonist, MK-801. Results indicate that MK-801 (0.5 mg/kg) significantly decreased social interaction following sub-chronic treatment (7 days). Treatment with PAOPA (1 mg/kg) blocked the effects of MK-801, and increased the amount of time spent in social interaction in comparison to control animals. These results provide evidence for the development of peptidomimetic compounds for the treatment of social withdrawal and related negative symptoms associated with schizophrenia.