Abdelaziz Mekhalfia

ATP analogues with modified phosphate chains and their selectivity for rat P2X2 and P2X2/3 receptors

Heteromeric P2X2/3 receptors are much more sensitive than homomeric P2X2 receptors to αβ‐methylene‐ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells. We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the αβ and/or βγ oxygen atoms were replaced by other moieties (including –CH2–, –CHF–, –CHCl–, –CHBr–, –CF2–, –CCl2–, –CBr2–, –CHSO3–, –CHPO3–, –CFPO3–, –CClPO3–, –CH2–CH2–, –C≡C–, –NH–, –CHCOOH–). We tested their actions as agonists or antagonists by whole‐cell recording from human embryonic kidney cells expressing P2X2 subunits alone (homomeric P2X2 receptors), or cells expressing both P2X2 and P2X3 subunits, in which the current through heteromeric P2X2/3 receptors was isolated. ADP analogues had no agonist or antagonist effect at either P2X2 or P2X2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X2 receptors, except βγ‐difluoromethylene‐ATP, which was a weak agonist. At P2X2/3 receptors, βγ‐imido‐ATP, βγ‐methylene‐ATP, and βγ‐acetylene‐ATP were weak agonists, whereas αβ,βγ‐ and βγ,γδ‐bismethylene‐AP4 were potent full agonists. βγ‐Carboxymethylene‐ATP and βγ‐chlorophosphonomethylene‐ATP were weak antagonists at P2X2/3 receptors (IC50 about 10 μM). The results indicate (a) that the homomeric P2X2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b) that the heteromeric P2X2/3 receptor is much more tolerant of αβ and βγ substitution; and (c) that a P2X2/3‐selective antagonist can be obtained by introduction of additional negativity at the βγ‐methylene.

Direct Screening for Phosphatase Activity by Turnover-Based Capture of Protein Catalysts

In studies towards the generation and identification of catalytic antibodies with phosphate monoesterase activity, we have developed a direct selection approach for covalent modification of catalysts founded on mechanism-based phosphatase inhibitors. It builds on the work of Halazy et al.[1] in the use of oand p-fluoromethylphenols to generate quinone methides as suicide substrates for glucosidases. Withers and co-workers conceived the use of 4-difluoromethylphenyl phosphate 1 as a suicide substrate for a human prostatic acid

HOMOCHIRAL SYNTHESIS OF AN AZA ANALOGUE OF S-ADENOSYL-L-METHIONINE (ADOMET) AND ITS BINDING TO THE E. COLI METHIONINE REPRESSOR PROTEIN (METJ)

A synthesis of 5′-{N-[(S)-3-amino-3-carboxypropyl]-methylamino}-5′-deoxyadenosine from D-adenosine and (S)-glutamic acid is described; this product, AzaAdoMet 2, has a pKa of 7.10 for the tertiary amino group and so acts as a charge-switchable analogue of AdoMet 1,a key component of polyamine biosynthesis; the binding of both 1 and 2 to the E. coli methionine repressor protein is investigated by X-ray crystallography.

Direkte Identifizierung von Proteinkatalysatoren mit Phosphatase‐Aktivität durch kovalente Bindung an ein Suizid‐Substrat

F r die Identifizierung und Herstellung katalytischer Antikˆrper mit Phosphomonoesterase-Aktivit‰t haben wir eine direkte Selektionsmethode etabliert, bei der, basierend auf Phosphatase-Suizid-Inhibitoren, die katalytischen Molek lspezies kovalent abgefangen wurden. Dem Konzept liegt die Arbeit von Halazy et al.[1] zugrunde, in der aus ound pFluormethylphenolen Chinonmethide als Suizid-Substrate f r Glucosidasen erzeugt werden. Withers et al. verwendeten 4-Difluormethylphenylphosphat 1 als Suizid-Substrat f r eine

Passive and catalytic antibodies and drug delivery

Antibodies are one of the most promising components of the biotechnology repertoire for the purpose of drug delivery. On the one hand, they are proven agents for cell-selective delivery of highly toxic agents in a small but expanding number of cases. This technology calls for the covalent attachment of the cytotoxin to a tumor-specific antibody by a linkage that is reversible under appropriate conditions (antibody conjugate therapy, ACT —“passive delivery”). On the other hand, the linker cleavage can be accomplished by a protein catalyst attached to the tumor-specific antibody (“catalytic delivery”). Where the catalyst is an enzyme, this approach is known as antibody-directed enzyme prodrug therapy (ADEPT). Where the transformation is brought about by a catalytic antibody, it has been termed antibody-directed abzyme prodrug therapy (ADAPT). These approaches will be illustrated with emphasis on how their demand for new biotechnology is being realized by structure-based protein engineering.