Mechanism of inhibition of the glutamate transporter EAAC1 by the conformationally constrained glutamate analogue (+)-HIP-B.

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-ß-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.

Protein engineering: a new frontier for biological therapeutics.

Protein engineering holds the potential to transform the metabolic drug landscape through the development of smart, stimulusresponsive drug systems. Protein therapeutics are a rapidly expanding segment of Food and Drug Administration approved drugs that will improve clinical outcomes over the long run. Engineering of protein therapeutics is still in its infancy, but recent general advances in protein engineering capabilities are being leveraged to yield improved control over both pharmacokinetics and pharmacodynamics. Stimulus- responsive protein therapeutics are drugs which have been designed to be metabolized under targeted conditions. Protein engineering is being utilized to develop tailored smart therapeutics with biochemical logic. This review focuses on applications of targeted drug neutralization, stimulus-responsive engineered protein prodrugs, and emerging multicomponent smart drug systems (e.g., antibody-drug conjugates, responsive engineered zymogens, prospective biochemical logic smart drug systems, drug buffers, and network medicine applications).