(±)-HIP-B

Mechanism of inhibition of the glutamate transporter EAAC1 by the conformationally constrained glutamate analogue (+)-HIP-B.[Pubmed:22703277]

Biochemistry. 2012 Jul 10;51(27):5486-95.

Glutamate transporters play an important role in the regulation of extracellular glutamate concentrations in the mammalian brain and are, thus, promising targets for therapeutics. Despite this importance, the development of pharmacological tools has mainly focused on the synthesis of competitive inhibitors, which are amino acid analogues that bind to the substrate binding site. In this report, we describe the characterization of the mechanism of glutamate transporter inhibition by a constrained, cyclic glutamate analogue, (+)-3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid [(+)-(3aS,6S,6aS)-HIP-B]. Our results show that (+)-HIP-B is a nontransportable amino acid that inhibits glutamate transporter function in a mixed mechanism. Although (+)-HIP-B inhibits the glutamate-associated anion conductance, it has no effect on the leak anion conductance, in contrast to competitive inhibitors. Furthermore, (+)-HIP-B is unable to alleviate the effect of the competitive inhibitor dl-threo-beta-benzyloxyaspartic acid (TBOA), which binds to the substrate binding site. (+)-HIP-B is more potent in inhibiting forward transport compared to reverse transport. In a mutant transporter, which is activated by glutamine, but not glutamate, (+)-HIP-B still acts as an inhibitor, although this mutant transporter is insensitive to TBOA. Finally, we analyzed the effect of (+)-HIP-B on the pre-steady-state kinetics of the glutamate transporter. The results can be explained with a mixed mechanism at a site that may be distinct from the substrate binding site, with a preference for the inward-facing configuration of the transporter and slow inhibitor binding. (+)-HIP-B may represent a new paradigm of glutamate transporter inhibition that is based on targeting of a regulatory site.

Dissociation of [3H]L-glutamate uptake from L-glutamate-induced [3H]D-aspartate release by 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid and 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid, two conformationally constrained aspartate and glutamate analogs.[Pubmed:15322243]

Mol Pharmacol. 2004 Sep;66(3):522-9.

We characterized the interaction of two conformationally constrained aspartate and glutamate analogs, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid (HIP-A) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B), with excitatory amino acid transporters (EAATs) in rat brain cortex synaptosomes. HIP-A and HIP-B were potent and noncompetitive inhibitors of [(3)H]L-glutamate uptake, with IC(50) values (17-18 microM) very similar to that of the potent EAAT inhibitor dl-threo-beta-benzyloxyaspartic acid (TBOA). The two compounds had little effect in inducing [(3)H]D-aspartate release from superfused synaptosomes but they potently inhibited l-glutamate-induced [(3)H]D-aspartate release, thus behaving as EAAT blockers, not substrates, in a manner similar to those of TBOA and dihydrokainate (DHK). HIP-A and HIP-B, but not TBOA and DHK, unexpectedly inhibited L-glutamate-induced [(3)H]D-aspartate release with IC(50) values (1.2-1.6 microM) 10 times lower than those required to inhibit [(3)H]L-glutamate uptake. There is therefore a concentration window (1-3 microM) in which the two compounds significantly inhibited l-glutamate-induced release with very little effect on L-glutamate uptake. This selective inhibitory effect required quite long preincubation (>5 min) of synaptosomes with the drugs. At these low concentrations, however, HIP-A and HIP-B had no effect on the EAAT-mediated [(3)H]d-aspartate release induced by altering the ion gradients, indicating that they specifically affect some L-glutamate-triggered process(es)--different from L-glutamate translocation itself--responsible for the induction of reverse transport. These data are inconsistent with the classic model of facilitated exchange-diffusion and provide the first evidence that EAAT-mediated substrate uptake and substrate-induced EAAT-mediated reverse transport are independent. Compounds such as HIP-A and HIP-B could be useful to further clarify the mechanisms underlying these operating modes of transporters.

Synthesis and enantiopharmacology of new AMPA-kainate receptor agonists.[Pubmed:10514280]

J Med Chem. 1999 Oct 7;42(20):4099-107.

Regioisomeric 3-carboxyisoxazolinyl prolines [CIP-A (+/-)-6 and CIP-B (+/-)-7] and 3-hydroxyisoxazolinyl prolines [(+/-)-8 and (+/-)-9] were synthesized and assayed for glutamate receptor activity. The tests were carried out in vitro by means of receptor binding techniques, second messenger assays, and the rat cortical wedge preparation. CIP-A showed a good affinity for both 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and kainic acid (KAIN) receptors. These results were confirmed in the cortical slice model where CIP-A displayed an EC(50) value very close to that of AMPA. The convulsant properties of all the compounds were evaluated in vivo on DBA/2 mice after icv injection. CIP-A showed a convulsant activity, measured as tonus and clonus seizures, 18-65 times higher than that produced by AMPA. It was also quite active after ip administration, since it induced seizures in mice at doses as low as 3.2 nmol/mouse. On the basis of the above-reported results we prepared and tested the enantiomers of CIP-A and CIP-B, obtained by reacting (S)-3,4-didehydroproline and (R)-3,4-didehydroproline, respectively, with ethoxycarbonylformonitrile oxide. In all the tests the S-form, CIP-AS [(-)-6], emerged as the eutomer evidencing common stereochemical requirements with the reference compounds AMPA and KAIN. Through modeling studies, carried out on CIP-A, AMPA, and KAIN, active conformations for CIP-AS and AMPA at AMPA receptors as well as for CIP-AS and KAIN at KAIN receptors are suggested.