Markéta Rinnová

Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N‐acetyl‐aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44–750) in Drosophila Schneiders cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha‐amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV‐like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac‐Glu‐Met, Ac‐Asp‐Met and, surprisingly, Ac‐Ala‐Glu and Ac‐Ala‐Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.

OLIGONUCLEOTIDES WITH ISOPOLAR PHOSPHONATE INTERNUCLEOTIDE LINKAGE: A NEW PERSPECTIVE FOR ANTISENSE COMPOUNDS?

Several types of isopolar modified oligothymidylates and oligoadenylates (15 mers) with the phosphonate -O-P-CH 2-O- internucleotide linkage were prepared. The modified oligonucleotides were subjected to the study of their hybridization properties, resistance against nucleases, and the ability to elicit RNase H activity.

Structural diversity of nucleoside phosphonic acids as a key factor in the discovery of potent inhibitors of rat T-cell lymphoma thymidine phosphorylase.

Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC(50) values below 1 microM were selected. Out of them, four compounds strongly inhibited the enzyme with IC(50) values lying in a range of 11-45 nM. These most potent compounds might be bi-substrate analogues.

Solid-phase peptide synthesis by fragment condensation: Coupling in swelling volume

The condensation of short peptides to resin-bound fragments was examined with respect to high coupling yields with only a small molar excess of a peptide in the reaction solution. The best results were achieved by the addition of reactants (C-unprotected peptide, DIC, and HOBt) dissolved in a so-called swelling volume of an appropriate solvent to a dry resin with an attached N-deprotected peptide chain. Each coupling step was followed by the end-capping of unreacted resin-bound peptide with 2,4-dinitrofluorobenzene. The substituted dinitroaniline chromophore formed in this reaction made the detection and separation of deletion peptides easy. Both conventional and ‘swelling volume’ methods were compared on parallel syntheses of the HIV-1 protease C-terminal 78–99 fragment. The yields of the isolated heneicosapeptide were 21 and 81% in favor of the ‘swelling volume’ procedure.

Isosteric phosphonate pyrrolidine-based dinucleoside monophosphate analogues.

Abstract A novel α- and β-configured pyrrolidine nucleoside phosphonates in adenine series were synthesized from trans-4-hydroxy-L-proline as starting material. d(ApA) analogues were also prepared and studied with respect to their hybridization properties with polyU.

Simultaneous multiple peptide synthesis: Comparison of T-bags and cotton

The methodology of SPPS has evolved over recent years into several approaches of SMPS. Many techniques of this type have been reported [1]. The increasing interest in cntton [2] as support for SPPS led us to perform a detailed comparison of the peptide synthesis on this carrier with one of the alternative multiple peptide synthesis approaches. We selected the combination of MeBHA resin and T-bags of Houghten [3].

Analysis of substrate specificity of HIV protease species

HIV protease (PR) is the most studied representative of all aspartic PRs. Soon after its identification, the HIV PR has been recognized as a prime target for rational drug design of anti-AIDS drugs. Although there are at present five compounds approved for clinical use as HIV PR inhibitors [1], the original urge for the design of a potent HIV PR inhibitor is not fading. Due to the rapid replication of the virus and high error rate of the reverse transcriptase, PR mutants evolve resistance towards clinically used drugs under the selection pressure of the inhibitors. Thus it is important to develop novel pharmaceutical leads, based on different chemistries, that might complement the available range of inhibitors and overcome the problem of viral resistance.