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PubMed1998

Limbic gamma rhythms. I. Phase-locked oscillations in hippocampal CA1 and subiculum.

Gamma oscillations (approximately 40 Hz) were induced in transverse hippocampal slices by tetanic stimulation of CA1 and/or subiculum. Tetanic stimulation of each site elicited population gamma oscillations in the surrounding tissue <400 micro(m) away. Stimulation of CA1 alone could evoke activity at both CA1 and subiculum. Subicular stimulation, however, did not transmit to CA1. When the rostral end of CA1 was stimulated, gamma oscillations transmitted across <1.5 mm of silent CA1 before reappearing in the subiculum. Tetanic stimulation of CA1 increased [K+]o to 8.2 +/- 1.5 mM (mean +/- SE). The location of the peak increase corresponded to the site of local gamma generation. Silent areas of CA1 experienced smaller [K+]o increases, to 4.9 +/- 0.7 mM. The subiculum, which generated gamma, remained at the baseline 3.0 mM. Although fluctuations in [K+]o may have an impact on the generation of gamma rhythms, they are not necessary for them. Gamma oscillations had similar frequencies in CA1 and subiculum (40.4 +/- 2.9 and 43.9 +/- 3.1 Hz, respectively). When present in both, the oscillations typically were phase locked with the subiculum lagging by 5.4 +/- 1.8 ms. When both CA1 and subiculum were stimulated the lag decreased by 28%. These delays approximate those expected for the conduction velocity of axons between the two regions, here estimated at 0.52 +/- 0.07 m/s. Transmission of gamma oscillations from CA1 to subiculum was blocked by the focal addition of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-receptor antagonist, 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione, to the subiculum. Oscillations induced in CA1 by local tetanic stimulation were blocked by focal application of the gamma-aminobutyric acid-A (GABAA) receptor antagonist, bicuculline, to CA1. Focal application of bicuculline to the subiculum blocked gamma due to subicular stimulation but not that due to CA1 stimulation. Bath-applied bicuculline disrupted subicular gamma evoked by subicular stimulation and led to a transient period of epileptiform responses before completely blocking responses. The further addition of the GABAB receptor antagonist, CGP 55845A, reversed this block, restoring the epileptic discharges evoked by tetanic stimulation. This suggests that the subiculum differs from hippocampal CA3 and neocortex, in having a powerful GABAB receptor-dependent mechanism to prevent epileptic discharges. The subiculum generates gamma rhythms both in response to local stimulation and to gamma rhythms evoked in CA1. Subicular gamma differs from that in CA1 in the presence of population spike doublets rather than singlets on many cycles. In both areas, generation of gamma by local stimulation depends on GABAA receptors, suggesting that the subiculum shares the interneuronal network mechanism we proposed for CA1.

Animals↗
PubMed2004

5-Hydroxytryptamine 1B receptors mediate presynaptic inhibition of monosynaptic IPSC in the rat dorsolateral septal nucleus.

Effects of 5-hydroxytryptamine (5-HT) on inhibitory synaptic transmission in the rat dorsolateral septal nucleus (DLSN) were examined by conventional intracellular and voltage-clamp recording methods. 5-HT (1-30 microM) depressed the monosynaptic fast IPSC evoked by local stimulation of the DLSN in the presence of DNQX, AP5 and CGP 55845A. CP 93129, a selective 5-HT1B receptor agonist, depressed the fast IPSC. The 5-HT-induced depression of the fast IPSC was attenuated by SB 216641, a selective antagonist for 5-HT1B receptors. 5-HT did not change the inward currents mediated by GABAA receptors, suggesting that 5-HT presynaptically inhibited the fast IPSC. 5-HT and CP 93129 depressed the frequency of miniature fast IPSPs (mIPSPs) without changing their amplitudes. Neither a selective protein kinase A inhibitor, H-89, nor a selective protein kinase C inhibitor, calphostin C, blocked the 5-HT-induced depression of the fast IPSC. N-Ethylmaleimide (NEM) blocked the 5-HT-induced depression of the evoked IPSC. These results suggest that activation of presynaptic 5-HT1B receptors depresses the release of GABA via a pertussis toxin (PTX)-sensitive G-protein in the rat DLSN.

Analysis of Variance↗
PubMed2004

Fast endocytosis is inhibited by GABA-mediated chloride influx at a presynaptic terminal.

Although multiple kinetic components of synaptic vesicle endocytosis have been identified, it has remained unclear whether neurons can differentially modulate these components. Using membrane capacitance measurements from isolated goldfish bipolar cell terminals, we found that the kinetics of endocytosis in retinal slices (single exponential decay; tau > 10 s) were significantly slower than those in acutely dissociated terminals (double exponential decay; tau(fast) approximately 1-2 s; tau(slow) > 10 s). Surprisingly, GABA(A) and/or GABA(C) receptor antagonists restored the fast component of endocytosis to terminals in retinal slices. Blocking GABAergic feedback from reciprocal synapses or removing external Cl(-) ions also allowed for fast endocytosis. Elevating internal Cl(-) via the patch pipette invariably slowed endocytosis, even in terminals dialyzed with increased Ca(2+) buffer. These results suggest a new role for GABA and Cl(-) ions in blocking the trigger for fast endocytosis at this ribbon-type synapse.

Animals↗
PubMed2006

Regulation of synaptic input to hypothalamic presympathetic neurons by GABA(B) receptors.

The hypothalamic paraventricular (PVN) neurons projecting to the spinal cord and brainstem play an important role in the control of homeostasis and the sympathetic nervous system. Although GABA(B) receptors are present in the PVN, their function in the control of synaptic inputs to PVN presympathetic neurons is not clear. Using retrograde tracing and whole-cell patch-clamp recordings in rat brain slices, we determined the role of presynaptic GABA(B) receptors in regulation of glutamatergic and GABAergic inputs to spinally projecting PVN neurons. The GABA(B) receptor agonist baclofen (1-50 microM) dose-dependently decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibitory postsynaptic currents (sIPSCs). The effect of baclofen on sEPSCs and sIPSCs was completely blocked by 10 microM CGP52432, a selective GABA(B) receptor antagonist. Baclofen also significantly reduced the frequency of both miniature excitatory and miniature inhibitory postsynaptic currents (mEPSCs and mIPSCs). Furthermore, uncoupling pertussis toxin-sensitive G(i/o) proteins with N-ethylmaleimide abolished baclofen-induced inhibition of mEPSCs and mIPSCs. However, the inhibitory effect of baclofen on the frequency of mIPSCs and mEPSCs persisted in the presence of either Cd2+, a voltage-gated Ca2+ channel blocker, or 4-aminopyridine, a blocker of voltage-gated K+ channels. Our results suggest that activation of presynaptic GABA(B) receptors inhibits synaptic GABA and glutamate release to PVN presympathetic neurons. This presynaptic action of GABA(B) receptors is mediated by the N-ethylmaleimide-sensitive G(i/o) proteins, but independent of voltage-gated Ca2+ and K+ channels.

4-Aminopyridine↗
PubMed2002

Molecular modeling of the interactions of glutamate carboxypeptidase II with its potent NAAG-based inhibitors.

Glutamate carboxypeptidase II (GCPII, NAALADase, or NAAG peptidase) is a catalytic zinc metallopeptidase. Its extracellular domain hydrolyzes the abundant neuropeptide, N-acetyl-L-aspartyl-L-glutamate (NAAG), to produce N-acetylaspartate and glutamate following the synaptic release of this transmitter. Thus, GCPII influences the extracellular concentrations of both glutamate and NAAG. NAAG activates group II metabotropic glutamate receptors, and activation of this receptor has been found to protect against anoxia-induced excitotoxic nerve cell death. In contrast, high levels of glutamate can be neurotoxic. Thus, GCPII is a potential therapeutic target for the reduction of excitotoxic levels of glutamate and enhancement of extracellular NAAG. To explore the structural basis of the interaction between GCPII and its inhibitors, we modeled the three-dimensional structure of the GCPII extracellular domain using a homology modeling approach. On the basis of the GCPII model, the structures of GCPII in complex with its potent inhibitors 2-(phosphonomethyl)pentanedioic acid (PMPA) and 4,4'-phosphinicobis(butane-1,3-dicarboxylic acid) (PBDA) were built by a computational docking method. The model of GCPII mainly consists of two alpha/beta/alpha sandwiches, between which two zinc ions are quadrivalently coordinated by the His379-Asp389-Asp455-H(2)O and the Asp389-Glu427-His555-H(2)O clusters, respectively. The ligand binding pocket is situated between these two sandwiches and is comprised of two subpockets: one is a surface-exposed highly positively charged subpocket; the other is a buried hydrophobic subpocket. The positively charged subpocket can accommodate the pharmacophore groups of inhibitor molecules (PMPA and PBDA) through the coordination of Zn(2+) with their phosphorus functionality and hydrogen-bonding interactions with Arg536, Arg538, and Ser456 (or Asn521), while the hydrophobic subpocket is engaged in hydrophobic and hydrogen-bonding interactions with the nonpharmacophore groups of PBDA. The predicted binding mode is consistent with the experimental data obtained from site-directed mutagenesis. On the basis of the predicted interaction mode, our structure-based design has led to a series of highly potent GCPII inhibitors.

Amino Acid Sequence↗
PubMed2005

Biosynthetic origins of C-P bond containing tripeptide K-26.

[reaction: see text] Primary metabolic precursors for K-26, a naturally occurring tripeptide phosphonic acid from Actinomyces sp. K-26, are investigated by heavy-atom isotope labeled substrate incorporation experiments. A highly sensitive selected reaction monitoring (SRM)-based method for isotopic incorporation estimation in natural products is reported. The incorporation of heavy-atom isotope labeled tyrosine compounds into the (R)-1-amino-2-(4-hydroxyphenyl)-ethylphosphonic acid moiety of compound K-26 suggests a new mechanism of biosynthesis of phosphonate functionality in natural products.

Actinomyces↗
PubMed2001

Kindling induces transient NMDA receptor-mediated facilitation of high-frequency input in the rat dentate gyrus.

To elucidate the gating mechanism of the epileptic dentate gyrus on seizure-like input, we investigated dentate gyrus field potentials and granule cell excitatory postsynaptic potentials (EPSPs) following high-frequency stimulation (10-100 Hz) of the lateral perforant path in an experimental model of temporal lobe epilepsy (i.e., kindled rats). Although control slices showed steady EPSP depression at frequencies greater than 20 Hz, slices taken from animals 48 h after the last seizure presented pronounced EPSP facilitation at 50 and 100 Hz, followed by steady depression. However, 28 days after kindling, the EPSP facilitation was no longer detectable. Using the specific N-methyl-D-aspartate (NMDA) and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproponic acid (AMPA) receptor antagonists 2-amino-5-phosphonovaleric acid and SYM 2206, we examined the time course of alterations in glutamate receptor-dependent synaptic currents that parallel transient EPSP facilitation. Forty-eight hours after kindling, the fractional AMPA and NMDA receptor-mediated excitatory postsynaptic current (EPSC) components shifted dramatically in favor of the NMDA receptor-mediated response. Four weeks after kindling, however, AMPA and NMDA receptor-mediated EPSCs reverted to control-like values. Although the granule cells of the dentate gyrus contain mRNA-encoding kainate receptors, neither single nor repetitive perforant path stimuli evoked kainate receptor-mediated EPSCs in control or in kindled rats. The enhanced excitability of the kindled dentate gyrus 48 h after the last seizure, as well as the breakdown of its gating function, appear to result from transiently enhanced NMDA receptor activation that provides significantly slower EPSC kinetics than those observed in control slices and in slices from kindled animals with a 28-day seizure-free interval. Therefore, NMDA receptors seem to play a critical role in the acute throughput of seizure activity and in the induction of the kindled state but not in the persistence of enhanced seizure susceptibility.

2-Amino-5-phosphonovalerate↗
PubMed2005

Enantioselective synthesis of nitroalkanes bearing all-carbon quaternary stereogenic centers through Cu-catalyzed asymmetric conjugate additions.

The first examples of catalytic asymmetric conjugate addition (ACA) of alkylzinc reagents to trisubstituted nitroalkenes, leading to the formation of nitroalkanes bearing a quaternary carbon stereogenic center, are reported. Reactions are promoted in the presence of 4 mol % of a readily available amino acid-based phosphine and 2 mol % (CuOTf).C6H6. Cu-catalyzed reactions proceed efficiently in up to 98% ee and can be carried out with a variety of dialkylzinc reagents and trisubstituted nitroolefins. We highlight the synthetic utility of the products obtained by efficient conversion of optically enriched nitroalkanes to the corresponding carboxylic acids.

Alkanes↗
PubMed1993

Echistatin disulfide bridges: selective reduction and linkage assignment.

Echistatin is the smallest member of the disintegrin family of snake venom proteins, containing four disulfides in a peptide chain of 49 residues. Partial assignment of disulfides has been made previously by NMR and chemical approaches. A full assignment was made by a newly developed chemical approach, using partial reduction with tris-(2-carboxyethyl)-phosphine at acid pH. Reduction proceeded in a stepwise manner at pH 3, and the intermediates were isolated by high performance liquid chromatography. Alkylation of free thiols, followed by sequencer analysis, enabled all four bridges to be identified: (1) at 20 degrees C a single bridge linking Cys 2-Cys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 degrees C the bridges Cys 7-Cys 32 and Cys 8-Cys 37 became accessible to the reagent and were reduced at approx. equal rates; (3) the two bicyclic peptides produced in this manner were less stable and could be reduced at 20 degrees C to a peptide that retains a single bridge linking Cys 20-Cys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 degrees C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2-11; 7-32; 8-37; 20-39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis.

Alkylation↗
PubMed2005

Intraglomerular inhibition: signaling mechanisms of an olfactory microcircuit.

Microcircuits composed of principal neuron and interneuron dendrites have an important role in shaping the representation of sensory information in the olfactory bulb. Here we establish the physiological features governing synaptic signaling in dendrodendritic microcircuits of olfactory bulb glomeruli. We show that dendritic gamma-aminobutyric acid (GABA) release from periglomerular neurons mediates inhibition of principal tufted cells, retrograde inhibition of sensory input and lateral signaling onto neighboring periglomerular cells. We find that L-type dendritic Ca(2+) spikes in periglomerular cells underlie dendrodendritic transmission by depolarizing periglomerular dendrites and activating P/Q type channels that trigger GABA release. Ca(2+) spikes in periglomerular cells are evoked by powerful excitatory inputs from a single principal cell, and glutamate release from the dendrites of single principal neurons activates a large ensemble of periglomerular cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2005

An NMDA receptor/nitric oxide cascade is involved in cerebellar LTD but is not localized to the parallel fiber terminal.

Long-term depression (LTD) of the parallel fiber-Purkinje cell synapse in the cerebellum is a cellular model system that has been suggested to underlie certain forms of motor learning. Induction of cerebellar LTD requires a postsynaptic kinase limb involving activation of mGluR1, protein kinase Calpha (PKCalpha), and phosphorylation of ser-880 on the AMPA receptor subunit GluR2. Several lines of evidence have also implicated a complementary phosphatase limb in which N-methyl-d-aspartate (NMDA) receptor-mediated Ca(2+) influx activates neuronal nitric oxide synthase (nNOS), the ultimate consequences of which are mediated by nitric oxide (NO), cGMP, and inhibition of postsynaptic protein phosphatases. However, the cellular localization of an NMDA/NO cascade has been complicated by the fact that neither functional NMDA receptors nor nNOS are expressed in Purkinje cells. This has lead to a proposal in which NMDA receptors activate nNOS in parallel fibers. Here, we confirm that pharmacological blockade of NMDA receptor or NO signaling blocks induction of LTD. However, no evidence was found for functional NMDA receptors in parallel fiber terminals: blockade of NMDA receptors did not alter either presynaptic Ca(2+) transients or the frequency of miniature excitatory postsynaptic currents. NMDA receptor blockade did abolish a slow depolarization evoked by burst stimulation of parallel fiber-stellate cell synapses. The application of NMDA evoked a Ca(2+) transient in stellate cell terminals but not in parallel fiber terminals. These results are consistent with the hypothesis that an NMDA receptor/NO cascade involved in cerebellar LTD is localized to interneurons rather than parallel fibers.

2-Amino-5-phosphonovalerate↗
PubMed2002

Selective inhibition of the C-domain of angiotensin I converting enzyme by bradykinin potentiating peptides.

Somatic angiotensin I converting enzyme (ACE) contains two functional active sites. Up to now, most of the studies aimed at characterizing the selectivity of inhibitors toward the two ACE active sites relied on the use of ACE mutants containing a single functional active site. By developing new fluorogenic synthetic substrates of ACE, we demonstrated that inhibitor selectivity can be assessed directly by using somatic ACE. This useful screening approach led us to discover that some bradykinin potentiating peptides turned out to be selective inhibitors of the C-domain of ACE. The peptide pGlu-Gly-Leu-Pro-Pro-Arg-Pro-Lys-Ile-Pro-Pro, with K(i)(app) values of 30 nM and 8 microM, respectively, for the C- and N-domain of ACE, is to our knowledge the most highly selective C-domain inhibitor of ACE so far reported. Inhibitors able to block selectively either the N- or C-domain of ACE will represent unique tools to probe the function of each domain in the regulation of blood pressure or other physiopathological events involving ACE activity.

Amino Acid Sequence↗
PubMed2005

Nicotine-induced switch in the nicotinic cholinergic mechanisms of facilitation of long-term potentiation induction.

Nicotine facilitates the induction of long-term potentiation (LTP) in the hippocampal CA1 region. The present study reveals the potential mechanisms underlying this effect of nicotine. Timed ACh-mediated activation of alpha7 nicotinic acetylcholine receptors (nAChRs) on pyramidal cells is known to promote LTP induction. Nicotine could suppress this timing-dependent mechanism by desensitizing nAChRs. Timed ACh-mediated activation of alpha7 nAChRs on feedforward interneurons can prevent LTP induction by inhibiting pyramidal cells. Nicotine diminished this ACh-mediated inhibition by desensitizing alpha7 nAChRs, thereby reducing the inhibitory influence on pyramidal cells. In addition to these desensitizing effects, nicotine activated presynaptic non-alpha7 nAChRs on feedforward interneurons to decrease the evoked release of gamma-aminobutyric acid (GABA) onto pyramidal cells. Furthermore, nicotine increased the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) in pyramidal cells, and concomitantly caused a reduction in the size of responses to focal GABA application onto the dendrites of pyramidal cells, suggesting that the nicotine-induced increase in interneuronal activity leads ultimately to a use-dependent depression of evoked IPSCs in pyramidal cells. These nicotine-induced suppressions of inhibition of pyramidal cells were accompanied by enhanced N-methyl-D-aspartate (NMDA) responses in pyramidal cells. Thus, our results suggest that nicotine promotes the induction of LTP by diminishing inhibitory influences on NMDA responses while suppressing the ACh-mediated mechanisms. These ACh-independent mechanisms probably contribute to the nicotine-induced cognitive enhancement observed in the presence of cholinergic deficits, such as those in Alzheimer's disease patients.

Acetylcholinesterase↗
PubMed1999

Long-term suppression of synaptic transmission by tetanization of a single pyramidal cell in the mouse hippocampus in vitro.

1. The consequences of stimulating a single pyramidal cell in the CA1 area of the hippocampus for synaptic transmission in the stratum radiatum were investigated. 2. Tetanic activation of single pyramids caused by depolarizing current injection, but not an equal number of distributed action potentials, reduced excitatory transmission by 20 %, with a delayed onset, for more than 1 h. 3. EPSPs in the tetanized pyramidal cells were increased for equally long periods but this was not the cause of the field EPSP reduction. Spontaneous somatic IPSPs were not affected; evoked IPSPs were decreased in the tetanized cell. 4. Paired pulse facilitation of the field EPSPs was unchanged. 5. The field EPSP reduction was markedly diminished by a knife cut along the base of pyramidal cells in CA1. 6. The addition of antagonists of GABA, NMDA and metabotropic glutamate receptors blocked or diminished the field EPSP slope reduction evoked by intracellular stimulation. 7. Simultaneous recordings revealed long-lasting excitations of interneurons located in the outer oriens layer as a result of single pyramid tetanization. 8. Intense firing of small numbers of pyramidal cells can thus persistently inhibit mass transmission through the hippocampus. This effect involves activation of interneurons by glutamate receptors.

2-Amino-5-phosphonovalerate↗
PubMed2000

GABAC receptors are localized with microtubule-associated protein 1B in mammalian cone photoreceptors.

Protein MAP1B was recently reported to link GABA(C) receptors to the cytoskeleton at neuronal synapses. This interaction was demonstrated in the mammalian retina, where GABA(C) receptors were thought to be exclusively expressed in bipolar cells. Our previous studies on cultured photoreceptors suggested however the presence of GABA(C) receptors in cones. To further investigate GABA(C) receptor expression in cones, we measured GABA responses in mammalian photoreceptors in situ, and we examined the distribution of the receptor and that of protein MAP1B in the mammalian outer retina. Photoreceptors were recorded from flat-mounted retinas of retinal degeneration mice at an age when the retina becomes cone-dominated after rod cell death. GABA(A) and GABA(C)-gated currents were produced only in cones but not rods. Recording freshly dissociated retinal cells from wild-type C57 mice confirmed the presence of GABA(A) and GABA(C) receptors in cones. Immunohistochemical labeling of mouse and rat retinal sections localized GABA(C) receptors to cone terminals that were identified by peanut agglutinin lectin staining. As expected from previous studies on bipolar cells, the punctate immunostaining was not restricted to cone terminals in the outer plexiform layer. MAP1B immunolabeling was obtained in rat and pig retinas and was similarly found in cone terminals identified by the peanut agglutinin lectin staining. These results provide physiological and histological evidence that cones receive a GABA feedback in the mammalian retina and are consistent with the notion that protein MAP1B links GABA(C) receptors to the cytoskeleton at postsynaptic sites.

Animals↗
PubMed2006

CGP 56999A, a GABA(B) receptor antagonist, enhances expression of brain-derived neurotrophic factor and attenuates dopamine depletion in the rat corpus striatum following a 6-hydroxydopamine lesion of the nigrostriatal pathway.

Rats were injected (i.p.) once daily with either 1 mg/kg CGP 56999A, a gamma-aminobutyric acid(B) (GABA(B)) receptor antagonist, or an equivalent volume of saline beginning 7 days prior to, and continuing for 7 days following, a unilateral 6-hydroxydopamine lesion of the nigrostriatal dopamine (DA) pathway. At the end of the CGP 56999A treatment period the concentrations of DA and dihydroxyphenylacetic acid (DOPAC), as well as the expression of brain-derived neurotrophic factor (BDNF), were analyzed in corpus striatum ipsilateral and contralateral to the lesioning. No significant differences in these parameters were noted in the contralateral striatum between saline- and CGP 56999A-treated subjects. In contrast, as compared to animals receiving saline only, daily treatment with the GABA(B) receptor antagonist significantly attenuated the 6-hydroxydopamine-induced decline in DA and increased the expression of BDNF in the ipsilateral striatum. The results indicate that CGP 56999A enhances BDNF gene expression in the rat corpus striatum and prevents the decline in DA content that is a characteristic sequela of 6-hydroxydopapmine lesions of the nigrostraital dopamine pathway. These findings suggest that GABA(B) receptor antagonists may be of value in the treatment of Parkinson's disease and other conditions that would benefit from an enhanced production of neurotrophic factors in brain.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2003

The most potent organophosphorus inhibitors of leucine aminopeptidase. Structure-based design, chemistry, and activity.

A new class of very potent inhibitors of cytosol leucine aminopeptidase (LAP), a member of the metalloprotease family, is described. The X-ray structure of bovine lens leucine aminopeptidase complexed with the phosphonic acid analogue of leucine (LeuP) was used for structure-based design of novel LAP inhibitors and for the analysis of their interactions with the enzyme binding site. The inhibitors were designed by modification of phosphonic group in the LeuP structure toward finding the substituents bound at the S' side of the enzyme. This resulted in two classes of compounds, the phosphonamidate and phosphinate dipeptide analogues, which were synthesized and evaluated as inhibitors of the enzyme. The in vitro kinetic studies for the phosphinate dipeptide analogues revealed that these compounds belong to the group of the most effective LAP inhibitors found so far. Their further modification at the P1 position resulted in more active inhibitors, hPheP[CH(2)]Phe and hPheP[CH(2)]Tyr (K(i) values 66 nM and 67 nM, respectively, for the mixture of four diastereomers). The binding affinities of these inhibitors toward the enzyme are the highest, if considering all compounds containing a phosphorus atom that mimic the transition state of the reaction catalyzed by LAP. To evaluate selectivity of the designed LAP inhibitors, additional tests toward aminopeptidase N (APN) were performed. The key feature, which determines their selectivity, is structure at the P1' position. Aromatic and aliphatic substituents placed at this position strongly interact with the LAP S1' binding pocket, while a significant increase in binding affinity toward APN was observed for compounds containing aromatic versus leucine side chains at the P1' position. The most selective inhibitor, hPheP[CH(2)]Leu, binds to LAP with 15 times higher affinity than to APN. One of the studied compounds, hPheP[CH(2)]Tyr, appeared to be very potent inhibitor of APN (K(i) = 36 nM for the mixture of four diastereomers). The most promising LAP inhibitors designed by computer-aided approach, the phosphonamidate dipeptide analogues, were unstable at pH below 12, because of the P-N bond decomposition, which excluded the possibility of determination of their binding affinities toward LAP.

Animals↗