Discovery and Synthesis of Hydroxy-l-Proline Blockers of the Neutral Amino Acid Transporters SLC1A4 (ASCT1) and SLC1A5 (ASCT2).

As a conformationally restricted amino acid, hydroxy-l-proline is a versatile scaffold for the synthesis of diverse multi-functionalized pyrrolidines for probing the ligand binding sites of biological targets. With the goal to develop new inhibitors of the widely expressed amino acid transporters SLC1A4 and SLC1A5 (also known as ASCT1 and ASCT2), we synthesized and functionally screened synthetic hydroxy-l-proline derivatives using electrophysiological and radiolabeled uptake methods against amino acid transporters from the SLC1, SLC7, and SLC38 solute carrier families. We have discovered a novel class of alkoxy hydroxy-pyrrolidine carboxylic acids (AHPCs) that act as selective high-affinity inhibitors of the SLC1 family neutral amino acid transporters SLC1A4 and SLC1A5. AHPCs were computationally docked into a homology model and assessed with respect to predicted molecular orientation and functional activity. The series of hydroxyproline analogs identified here represent promising new agents to pharmacologically modulate SLC1A4 and SLC1A5 amino acid exchangers which are implicated in numerous pathophysiological processes such as cancer and neurological diseases.

Synthesis and preliminary pharmacological evaluation of novel derivatives of L-beta-threo-benzylaspartate as inhibitors of the neuronal glutamate transporter EAAT3.

A series of beta-benzylaspartate derivatives were prepared from N-trityl-L-aspartate dimethyl ester and evaluated as inhibitors of neuronal glutamate transporter EAAT3. The result of the structure-activity studies suggests that the position occupied by the aromatic ring of beta-benzylaspartate within the binding site of EAAT3 may be different from that occupied by comparable groups in previously identified inhibitors, such as L-threo-benzyloxy aspartate (TBOA). Further, halogen substitutions at the 3-position of the aromatic ring of beta-benzylaspartate can increase the potency with which the analogues inhibit EAAT3.

Design, synthesis, and biological activity of novel triazole amino acids used to probe binding interactions between ligand and neutral amino acid transport protein SN1.

Novel triazole amino acids were synthesized as probes to investigate ligand-protein binding interactions of the neutral amino acid transporter SN1. The bonding hypothesis to be tested was that the side chains of endogenous substrates are acting as H-bond acceptors. Although limited inhibition of (3)H-L-glutamine uptake by SN1 expressing oocytes was observed, the synthetic compounds show a trend that suggests a hydrogen bond interaction just outside the endogenous ligand binding pocket.

The substituted aspartate analogue L-beta-threo-benzyl-aspartate preferentially inhibits the neuronal excitatory amino acid transporter EAAT3.

The excitatory amino acid transporters (EAATs) play key roles in the regulation of CNS L-glutamate, especially related to synthesis, signal termination, synaptic spillover, and excitotoxic protection. Inhibitors available to delineate EAAT pharmacology and function are essentially limited to those that non-selectively block all EAATs or those that exhibit a substantial preference for EAAT2. Thus, it is difficult to selectively study the other subtypes, particularly EAAT1 and EAAT3. Structure activity studies on a series of beta-substituted aspartate analogues identify L-beta-benzyl-aspartate (L-beta-BA) as among the first blockers that potently and preferentially inhibits the neuronal EAAT3 subtype. Kinetic analysis of D-[(3)H]aspartate uptake into C17.2 cells expressing the hEAATs demonstrate that L-beta-threo-BA is the more potent diastereomer, acts competitively, and exhibits a 10-fold preference for EAAT3 compared to EAAT1 and EAAT2. Electrophysiological recordings of EAAT-mediated currents in Xenopus oocytes identify L-beta-BA as a non-substrate inhibitor. Analyzing L-beta-threo-BA within the context of a novel EAAT2 pharmacophore model suggests: (1) a highly conserved positioning of the electrostatic carboxyl and amino groups; (2) nearby regions that accommodate select structural modifications (cyclopropyl rings, methyl groups, oxygen atoms); and (3) a unique region L-beta-threo-BA occupied by the benzyl moieties of L-TBOA, L-beta-threo-BA and related analogues. It is plausible that the preference of L-beta-threo-BA and L-TBOA for EAAT3 and EAAT2, respectively, could reside in the latter two pharmacophore regions.

The excitatory amino acid transporters: pharmacological insights on substrate and inhibitor specificity of the EAAT subtypes.

L-glutamate serves as the primary excitatory neurotransmitter in the mammalian CNS, where it can contribute to either neuronal communication or neuropathological damage through the activation of a wide variety of excitatory amino acid (EAA) receptors. By regulating the levels of extracellular L-glutamate that have access to these receptors, glutamate uptake systems hold the potential to effect both normal synaptic signaling and the abnormal over-activation of the receptors that can trigger excitotoxic pathology. Among the various membrane transporters that are capable of translocating this dicarboxylic amino acid, the majority of glutamate transport in the CNS, particularly as related to excitatory transmission, is mediated by the high-affinity, sodium-dependent, excitatory amino acid transporters (EAATs). At least 5 subtypes of EAATs have been identified, each of which exhibits a distinct distribution and pharmacology. Our growing appreciation for the functional significance of the EAATs is closely linked to our understanding of their pharmacology and the consequent development of inhibitors and substrates with which to delineate their activity. As was the case with EAA receptors, conformationally constrained glutamate mimics have been especially valuable in this effort. The success of these compounds is based upon the concept that restricting the spatial positions that can be occupied by required functional groups can serve to enhance both the potency and selectivity of the analogues. In the instance of the transporters, useful pharmacological probes have emerged through the introduction of additional functional groups (e.g., methyl, hydroxyl, benzyloxy) onto the acyclic backbone of glutamate and aspartate, as well as through the exploitation of novel ring systems (e.g., pyrrolidine-, cyclopropyl-, azole-, oxazole-, and oxazoline-based analogues) to conformationally lock the position of the amino and carboxyl groups. The focus of the present review is on the pharmacology of the EAATs and, in particular, the potential to identify those chemical properties that differentiate the processes of binding and translocation (i.e., substrates from non-substrate inhibitors), as well as strategies to develop glutamate analogues that act selectively among the various EAAT subtypes.

Ngamma-aryl glutamine analogues as probes of the ASCT2 neutral amino acid transporter binding site.

Analogues of L-glutamine were designed and synthesized to test a hydrogen-bond hypothesis between ligand and neutral amino acid transporter ASCT2. The key design feature contains a substituted phenyl ring on the amide nitrogen that contains electron withdrawing and electron donating groups that alter the pKa of the amide NH. Through this study a preliminary binding site map has been developed, and a potent commercially available competitive inhibitor of the ASCT2 transporter has been identified.

Synthesis and in vitro pharmacology of substituted quinoline-2,4-dicarboxylic acids as inhibitors of vesicular glutamate transport.

The vesicular glutamate transport (VGLUT) system selectively mediates the uptake of L-glutamate into synaptic vesicles. Uptake is linked to an H+-ATPase that provides coupling among ATP hydrolysis, an electrochemical proton gradient, and glutamate transport. Substituted quinoline-2,4-dicarboxylic acids (QDCs), prepared by condensation of dimethyl ketoglutaconate (DKG) with substituted anilines and subsequent hydrolysis, were investigated as potential VGLUT inhibitors in synaptic vesicles. A brief panel of substituted QDCs was previously reported (Carrigan et al. Bioorg. Med. Chem. Lett. 1999, 9, 2607-2612)(1) and showed that certain substituents led to more potent competitive inhibitors of VGLUT. Using these compounds as leads, an expanded series of QDC analogues were prepared either by condensation of DKG with novel anilines or via aryl-coupling (Suzuki or Heck) to dimethyl 6-bromoquinolinedicarboxylate. From the panel of almost 50 substituted QDCs tested as inhibitors of the VGLUT system, the 6-PhCH=CH-QDC (K(i) = 167 microM), 6-PhCH2CH2-QDC (K(i) = 143 microM), 6-(4'-phenylstyryl)-QDC (K(i) = 64 microM), and 6-biphenyl-4-yl-QDC (K(i) = 41 microM) were found to be the most potent blockers. A preliminary assessment of the key elements needed for binding to the VGLUT protein based on the structure-activity relationships for the panel of substituted QDCs is discussed herein. The substituted QDCs represent the first synthetically derived VGLUT inhibitors and are promising templates for the development of selective transporter inhibitors.