Reduction Of Inhibitory Potency Research Articles

Transmembrane AMPA receptor regulatory protein regulation of competitive antagonism: a problem of interpretation

Synaptic AMPA receptors are greatly influenced by a family of transmembrane AMPA receptor regulatory proteins (TARPs) which control trafficking, channel gating and pharmacology. The prototypical TARP, stargazin (or γ2), shifts the blocking ability of several AMPAR-selective compounds including the commonly used quinoxalinedione antagonists, CNQX and NBQX. Stargazin's effect on CNQX is particularly intriguing as it not only apparently lowers the potency of block, as with NBQX, but also renders it a partial agonist. Given this, agonist behaviour by CNQX has been speculated to account for its weaker blocking effect on AMPAR-TARP complexes. Here we show that this is not the case. The apparent effect of stargazin on CNQX antagonism can be almost entirely explained by an increase in the apparent affinity for l-glutamate (l-Glu), a full agonist and neurotransmitter at AMPAR synapses. Partial agonism at best plays a minor role but not through channel gating per se but rather because CNQX elicits AMPAR desensitization. Our study reveals that CNQX is best thought of as a non-competitive antagonist at glutamatergic synapses due to the predominance of non-equilibrium conditions. Consequently, CNQX primarily reports the proportion of AMPARs available for activation but may also impose additional block by receptor desensitization.

FMRFamides exert a unique modulation of rodent pancreatic polypeptide sensitive neuropeptide Y (NPY) receptors

FMRFamide and related peptides (RFamides) were found to inhibit the association binding of iodinated human pancreatic polypeptide ([125I]hPP) to Y5-like neuropeptide Y (NPY) receptor in rodent tissues. An allosteric regulation of the activity of the rodent kidney PP-sensitive neuropeptide Y (NPY) receptor by RFamides was indicated by potency decrease with particle concentration in the inhibition of the association binding of 125I-labeled human pancreatic polypeptide (hPP) by RFamides at rabbit kidney membranes. The competition by C-terminal hexapeptide of hPP (LTRPRY.NH2) did not show such affinity change. The steady-state binding of hPP showed little sensitivity to any of the RFamides tested. The Y1-selective binding of [125I][Leu31,Pro34]hPYY (at 2 nM hPP) was much less sensitive to RFamides than the binding of [125I]hPP, albeit with some differences across tissue or cell types. The binding of Y2-selective agonist 125I-labeled human peptide YY (3-36) was quite insensitive to RFamides. The presence of a unique component in the inhibition of hPP binding by RFamides was further indicated by a degree of antagonism with phospholipase C inhibitor U-73122, and by an only limited cooperation with a N5-amiloride compound, and with alkylator chloroethylclonidine. Change of the chirality of individual residues in the FMRFamide molecule produced a significant reduction of inhibitory potency only with D-Phe in the C-terminal position. Substitution of the (C-3) L-Met by L-Leu greatly increased the inhibitory potency of RFamides relative to otherwise identical congeners. RFamides could act both as ligands of membrane neighbors of the PP receptor, and as competitors of Y5-like NPY receptor epitopes that accommodate the C-terminal aspects of agonist peptides.

Retention of Inhibitory Potency of an ACE Inhibitor Conjugated with Rh(III) and Pd(II) (Iminophosphorano)phosphines. Synthesis and X-ray Structural Investigations

Succinimido, amido, and ester functionalized tetrafluoroaryl azides selectively oxidize bisdiphenylphosphinomethane at one of the P(III) centers giving (iminophosphorano)phosphines 6, 7, and 8 respectively in high yields. Succinimido functionalized perfluoroaryl azido (iminophosphorano)phosphine is attached to angiotensin converting enzyme (ACE) inhibitor, lisinopril at one end, leaving the other end for chelation to Rh(III) and Pd(II) precursors including radioactive analogues establishing the hetero-bifunctionality for potential in vivo tracking of the radiotracer. The measurement of inhibitory potency of lisinopril−metal conjugates (Rh and Pd), modified through the primary amine reveals an increase in inhibitory potency, although small, retaining the target potential of native lisinopril toward specific biological sites. However, direct complexation utilizing the carboxylic groups of lisinopril with a Cu precursor resulted in the reduction of inhibitory potency from nM to μM levels rendering it less us...

Synthetic strategies to lower affinity for CYP2D6.

There are several models for the CYP2D6 active site with the characteristics of their substrates and inhibitors well defined. Imipramine possesses such characteristics and is both a substrate and an inhibitor of the CYP2D6 enzyme. Possible synthetic strategies to avoid interaction with the enzyme have been investigated, including: attenuation of basicity; and alteration of rigidity and length of the alkyl chain. Imipramine inhibited the 1'-hydroxylation of bufuralol (10 microM), an in vitro marker of CYP2D6 activity, in a CYP2D6 cell line (IC50 = 2.4 microM). Inhibitory potency was attenuated by the removal of the basic centre; imipramine N-oxide had no inhibitory effect on bufuralol 1'-hydroxylation. However, removal of this basic centre, as a strategy to decrease CYP2D6 interaction, may well have a detrimental effect on pharmacological efficacy. Both an increase and decrease in the N-N carbon chain length [2C,4C] caused a reduction in inhibitory potency. In addition, introduction of a carbonyl adjacent to the amino dibenzyl moiety into 2C, 3C and 4C compounds brought about a further reduction in inhibitory potency. These data demonstrate that changes to the molecule, distal to the basic centre, can attenuate the affinity of the molecule for CYP2D6 and are in keeping with the known characteristics of the enzyme.

Identification of Critical Determinants for Autoinhibition in the Pseudosubstrate Region of Type Iα cAMP-dependent Protein Kinase

The consensus substrate site for cAMP-dependent protein kinase (PKA) is Arg-Arg-Xaa-Ser(P)-Xaa and the autoinhibitory domain of the PKA type I alpha regulatory subunit (RI subunit) contains a similar sequence, Arg92-Arg-Arg-Arg-Gly-Ala-Ile-Ser-Ala-Glu. The italicized amino acids form a putative pseudosubstrate site (Ser is replaced with Ala), which together with adjacent residues could competitively inhibit substrate phosphorylation by the PKA catalytic subunit (C subunit). The present studies determine the contributions of Arg92-95, Ile98, and Glu101 to inhibitory potency. Amino-terminal truncation of RI subunit through Arg92 (delta1-92) or Arg93 (delta1-93) had no detectable effect on inhibition of C subunit. Truncation through Arg94 (delta1-94), or point mutation of Arg95 within truncated mutants (delta1-93.R95A or delta1-92.R95A), caused a dramatic reduction in inhibitory potency. Truncation through Arg95 (delta1-95) had a greater effect than did replacement or deletion of Arg94 or Arg95 alone. Using full-length RI subunit, the inhibitory potency was reduced by replacing Ile98 with Ala, Gly, or Gln, but not by replacing it with Val. The inhibitory potency of RI subunit was unchanged when Glu101 was replaced with Ala or Gln. It is concluded that Arg94, Arg95 and, to a lesser extent, Ile98 are vital constituents of PKA autoinhibition by type I alpha R subunit.

Reabsorption of dicarboxylic acids from the proximal convolution of rat kidney.

The transport of dicarboxylic acid in the late proximal convolution was investigated by measuring the 3.5 s efflux of 2-oxoglutarate from the tubular lumen of rats starved for 3 days. The 3.5 s efflux of 2-oxoglutarate comprises two components, one due to movement across the brush border, obeying Michaelis Menten kinetics with an apparent Km of 0.13 mmol/l and a Jmax of 0.41 pmol cm-1 s-1 and the other due to diffusional movement presumably via the paracellular pathway with a permeability of 23.0 microns2 s-1. Omission of sodium from the perfusion fluid reduced the transcellular efflux of 2-oxoglutarate by 76%, indicating a sodium-dependent transport system. Addition of 5 mmol/l lithium to the liminal and capillary perfusate reduced it by 56% indicating a specific inhibitory effect of lithium on dicarboxylic acid transport. Addition of 5 mmol/l H2DIDS to the luminal perfusate reduced 3.5 s transcellular 2-oxoglutarate efflux by 35%. The molecular specificity of the system was assessed by studying the inhibitory effects of a series of dicarboxylates, both aliphatic and aromatic, on the 3.5 s efflux of 2-oxoglutarate. Inhibitory constants (apparent Ki) were calculated for comparative purposes assuming competitive inhibition. From this, the system was found to have optimal affinity for dicarboxylates in the trans-configuration with a four or five carbon chain (i.e. succinate and glutarate). Substitution on the 2-carbon atom with CH3-, OH-, SH-, and O = resulted in little reduction in inhibitory potency as compared to succinate itself. However, 2-substitution with NH3+ (not with N-acetyl) as well as 2,3-disubstitution with CH3-, OH- or SH-, strongly reduced or abolished the inhibitory potency. Only with the exception of pyruvate all monocarboxylates tested, did not inhibit 2-oxoglutarate transport, but all tricarboxylates tested, i.e. citrate, isocitrate and tricarballate had an inhibitory effect. Citrate inhibition was higher at acidic than alkaline pH. A number of aromatic compounds was also tested. In most cases the inhibitory potency of the aromatic compounds was considerably weaker than that of the effective 4-5 carbon chain aliphatic compounds. Only benzene-1,4-dicarboxylate, benzene-2-nitro-1,4-dicarboxylate, and benzene-1,2-diacetate had a high inhibitory potency. In the case of the aromatic dicarboxylates the most important feature relating molecular structure to transport was the distance between the two carboxyl-groups in the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)