Biomedical subjects

Robert Esnouf

Publications and source records attributed to Robert Esnouf.

7 recordsLinked to original sources

PubMed2005

Design of a data model for developing laboratory information management and analysis systems for protein production.

Data management has emerged as one of the central issues in the high-throughput processes of taking a protein target sequence through to a protein sample. To simplify this task, and following extensive consultation with the international structural genomics community, we describe here a model of the data related to protein production. The model is suitable for both large and small facilities for use in tracking samples, experiments, and results through the many procedures involved. The model is described in Unified Modeling Language (UML). In addition, we present relational database schemas derived from the UML. These relational schemas are already in use in a number of data management projects.

Algorithms↗

PubMed2005

Image storage for automated crystallization imaging systems.

To use crystallography for the determination of the three-dimensional structures of proteins, protein crystals need to be grown. Automated imaging systems are increasingly being used to monitor these crystallization experiments. These present problems of accessibility to the data, repeatability of any image analysis performed and the amount of storage required. Various image formats and techniques can be combined to provide effective solutions to high volume processing problems such as these, however lack of widespread support for the most effective algorithms, such as JPeg2000 which yielded a 64% improvement in file size over the bitmap, currently inhibits the immediate take up of this approach.

Algorithms↗

PubMed2004

Interaction of cycloSal-pronucleotides with cholinesterases from different origins. A structure-activity relationship.

A large number of cycloSal-nucleotide triesters 1-49 have been studied concerning their ability to inhibit cholinesterases of different origins as well as to inhibit HIV replication in cell culture. It was shown that none of the triesters showed inhibitory effects against human acetylcholinesterase (AChE; isolated enzyme) as well as against AChE from beef erythrocytes and calf serum. In contrast, inhibition of butyrylcholinesterase (BChE) has been observed for some triesters in human and mouse serum. cycloSal pronucleotides showed strong competitive inhibition with respect to the substrate acetylcholine chloride (K(i)/K(m): approximately 2 x 10(-5)) and acted by time-dependent irreversible inhibition of the human serum BChE. Detailed studies demonstrated that the inhibitory effect against BChE is dependent on the nucleoside analogue, the substitution pattern of the cycloSal-moiety, and particularly on the stereochemistry at the phosphorus atom. Structural requirements to avoid the inhibition of BChE by cycloSal-nucleotide triesters have been elucidated in the reported study.

Acetylcholinesterase↗

PubMed2004

Susceptibility of feline immunodeficiency virus/human immunodeficiency virus type 1 reverse transcriptase chimeras to non-nucleoside RT inhibitors.

To map the determinants of the lack of susceptibility of feline immunodeficiency virus (FIV) reverse transcriptase (RT) to anti human immunodeficiency virus type 1 (HIV-1) non-nucleoside RT inhibitors (NNRTIs), a variety of chimeric HIV-1/FIV RTs were constructed. The majority of chimeric RTs had an affinity (Km) for their natural substrates comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy was decreased. Whereas HIV-1 RT could be made entirely insensitive to NNRTIs by exchanging the amino acid sequence 97 through 205 of FIV RT, none of the reverse FIV/HIV-1 RT chimeras gained susceptibility to NNRTIs. The amino acids that are thought to be involved in NNRTI susceptibility and that are different from those in HIV-1 RT have also been introduced in FIV RT. These mutant RTs gained virtually no susceptibility to efavirenz or capravirine. Vice versa, when these HIV-1-specific amino acids were replaced by their FIV RT counterparts in HIV-1 RT, susceptibility to the NNRTIs was lost. Thus, replacing segments or substituting relevant amino acids in FIV RT by their HIV-1 RT counterparts did not suffice to make FIV RT sensitive toward NNRTIs and was often accompanied by a decrease or even total loss of polymerase activity. It is postulated that, in contrast to the results found for HIV-1/HIV-2 RT chimeras and supported by the crystal structure of HIV-2 RT, there exist significant differences in the structure and/or flexibility of FIV RTs that may prevent NNRTIs from interacting with the FIV RT.

Amino Acid Sequence↗

PubMed2003

Non-nucleoside inhibitors of mitochondrial thymidine kinase (TK-2) differentially inhibit the closely related herpes simplex virus type 1 TK and Drosophila melanogaster multifunctional deoxynucleoside kinase.

5'-O-Trityl derivatives of thymidine (dThd), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), and their acyclic analogs 1-[(Z)-4-triphenylmethoxy-2-butenyl]thymine (KIN-12) and (E)-5-(2-bromovinyl)-1-[(Z)-4-triphenylmethoxy-2-butenyl]uracil (KIN-52) have been synthesized and evaluated for their inhibitory activity against the amino acid sequence related mitochondrial dThd kinase (TK-2), herpes simplex virus type 1 (HSV-1) TK, and Drosophila melanogaster multifunctional 2'-deoxynucleoside kinase (Dm-dNK). Several compounds proved markedly inhibitory to these enzymes and represent a new generation of nucleoside kinase inhibitors. KIN-52 was the most potent and selective inhibitor of TK-2 (IC(50), 1.3 microM; K(i), 0.50 microM; K(i)/K(m), 0.37) but was not inhibitory against HSV-1 TK and Dm-dNK at 100 microM. As found for the alternative substrate BVDU, the tritylated compounds competitively inhibited the three enzymes with respect to dThd. However, whereas BVDU behaved as a noncompetitive inhibitor (alternative substrate) of TK-2 and HSV-1 TK with respect to ATP as the varying substrate, the novel tritylated enzyme inhibitors emerged as reversible purely uncompetitive inhibitors of these enzymes. Computer-assisted modeling studies are in agreement with these findings. The tritylated compounds do not act as alternative substrates and they showed a type of kinetics against the nucleoside kinases different from that of BVDU. KIN-12, and particularly KIN-52, are the very first non-nucleoside specific inhibitors of TK-2 reported and may be useful for studying the physiological role of the mitochondrial TK-2 enzyme.

Adenosine Triphosphate↗

PubMed2002

The A167Y mutation converts the herpes simplex virus type 1 thymidine kinase into a guanosine analogue kinase.

The thymidine (dThd) kinase (TK) encoded by herpes simplex virus type 1 (HSV-1) is not only endowed with dThd kinase, but also with thymidylate (dTMP) kinase and 2'-deoxycytidine (dCyd) kinase (dCK) activity. HSV-1 TK also recognizes a variety of antiherpetic guanine nucleoside analogues such as acyclovir (ACV), ganciclovir (GCV), lobucavir (LBV), penciclovir (PCV), and others (i.e., A5021). Site-directed mutagenesis of the highly conserved Ala-167 to Tyr in HSV-1 TK completely abolished TK, dTMP-K, and dCK activity, but maintained ACV-, GCV-, LBV-, PCV-, and A5021-phosphorylating capacity. A variety of 5-substituted pyrimidine nucleoside substrates, but also a number of selective HSV-1 TK inhibitors structurally related to thymine lost significant binding affinity for the mutant enzyme and did not markedly compete with GCV phosphorylation by the mutant enzyme. These findings could be explained by computer-assisted modeling data that revealed steric hindrance of the pyrimidine ring in the HSV-1 TK active site by the large 4-hydroxybenzyl ring of 167-Tyr, while the positioning of the purine ring of guanine-based HIV-1 TK substrates in the active site was kept virtually unaltered. Surprisingly, the efficiency of conversion the antiherpetic 2'-deoxyguanosine analogues ACV, GCV, LBV, PCV, and A5021 to their phosphorylated forms by the A167Y mutant HSV-1 TK was far more pronounced than for the wild-type enzyme. Therefore, the single A167Y mutation converts the wild-type HSV-1 TK from a predominantly pyrimidine nucleos(t)ide kinase into a virtually exclusive purine (guanine) nucleoside analogue kinase.

Alanine↗

PubMed2002

Lack of susceptibility of bicyclic nucleoside analogs, highly potent inhibitors of varicella-zoster virus, to the catabolic action of thymidine phosphorylase and dihydropyrimidine dehydrogenase.

The susceptibility of the bicyclic nucleoside analogs (BCNAs), highly potent and selective inhibitors of varicella-zoster virus (VZV), to the enzymes involved in nucleoside/nucleobase catabolism has been investigated in comparison with the established anti-VZV agent (E)-5-(2-bromovinyl)-2'-deoxyuridine [BVDU; brivudine (Zostex)]. Whereas human and bacterial thymidine phosphorylases (TPases) efficiently converted BVDU to its antivirally inactive free base (E)-5-(2-bromovinyl)uracil (BVU), BCNAs showed no evidence of conversion to the free base in the presence of these enzymes. The lack of substrate affinity of TPase for the BCNAs could be rationalized by computer-assisted molecular modeling of the BCNAs in the TPase active site. Moreover, in contrast with BVU, which is a potent and selective inhibitor of dihydropyrimidine dehydrogenase (DPD) (50% inhibitory concentration; 10 microM in the presence of a 25 microM concentration of the natural substrate thymine), the free base (Cf 1381; 6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) of BCNA (Cf 1368; 3-(2'-deoxy-beta-D-ribofuranosyl)-6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) and the free base Cf 2200 [6-(4-n-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one] of BCNA (Cf 1743; 3-(2'-deoxy-beta-D-ribofuranosyl)-6-(4-n-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) did not inhibit the DPD-catalyzed catabolic reaction of pyrimidine bases (i.e., thymine) and pyrimidine base analogs [i.e., 5-fluorouracil (FU)] at a concentration of 250 microM. Consequently, whereas BVU caused a dramatic rise of FU levels in FU-treated mice, the BCNAs did not affect FU levels in such mice. From our data it is evident that BCNAs represent highly stable anti-VZV compounds that are not susceptible to breakdown by nucleoside/nucleobase catabolic enzymes and are not expected to interfere with cellular catabolic processes such as those involved in FU catabolism.

Animals↗