ISSRNS 2012: Abstracts / Synchrotron Radiation in Natural Science Vol. 11, No 1 – 2 (2012) P 11
SEARCHING FOR THE DIFFERENCES BETWEEN TRICHINELLA SPIRALIS AND MOUSE THYMIDYLATE SYNTHASES:
A QUEST FOR SPECIES-SPECIFIC DRUGS
A. Dowiercia l1∗, A. Jarmu la1, W.R. Rypniewski2, T. Fr¸aczyk1, P. Wilk1, and W. Rode1
1Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Str., 02–093 Warszawa, Poland
2Institute of Bioorganic Chemistry, Polish Academy of Sciences, 12/14 Noskowskiego Str., 61–704 Pozna´n, Poland
Keywords: thymidylate synthase, Trichinella spiralis, parasitic nematode, selective inhibitor, synchrotron radiation
∗e-mail : a.dowiercial@nencki.gov.pl
The enzyme thymidylate synthase [TS: 2.1.1.45]
catalyzes the multi-step reaction of methylation of dUMP, leading to dTMP, in the presence of the cofactor, N5,10-methylenetetrahydrofolate. As indispensable component in all known proliferat- ing systems, thymidylate synthase is an impor- tant molecular target for anticancer, antifungal, an- tivirus and antibacterial drugs.
Trichinella spiralis infection is responsible for trichinellosis, a serious disease, occurring regularly also in Poland. In view of high thymidylate synthase activity found throughout the parasite developmen- tal cycle, the enzyme appears a potential target for chemotherapeutic drugs [1].
TS is one of the most conservative enzymes known, with the amino acid sequence identity for human and E.coli TSs’ being 51% and similarity amounting to 67%; the corresponding values for the human and mouse enzymes are 88% and 93%, re- spectively. The high sequence similarity between TSs’ of different origin results in a high degree of similarity of their spatial structures, as confirmed by large numbers of TS crystal structures deposited in the PDB database.
In view of this close structural similarity of dif- ferent TS’s, and particularly their active centers, inhibitors designed as substrate/cofactor analogues are beyond hope as candidates for species-selective inhibitors of the pathogen vs. mammalian enzyme.
A possible solution, successfully applied to search for a specific inhibitor of bacterial vs. human TS [2], is virtual selection of an inhibitor, based on com- parison of the 3D structures of pathogen and human
enzyme proteins, aimed at non-conservative protein fragments differing between both proteins.
In order to take advantage of such an approach, we made an attempt to find structural differences between TS of the parasitic nematode Trichinella spiralis and the mammalian enzyme. It is worth- while to mention that thus far PDB does not contain any model of invertebrate TS.
Therefore we undertook crystallization of Trichinella spiralis TS complexed with the sub- strate, dUMP. Having obtained suitable monocrys- tals, we collected the diffraction data to the 1.9 ˚A resolution using synchrotron radiation at BESSY in Berlin. Preliminary analysis of solved structure of the parasitic nematode thymidylate synthase, com- pared to the mouse enzyme, points to at least two structural differences.
Acknowledgments: This work was supported by the National Science Centre Grant No.
2011/01/B/NZ6/01781.
References
[1] M. D¸abrowska, E. Jagielska, J. Cie´sla, A. P lucienniczak, J. Kwiatkowski, M. Wranicz, P. Boireau, W. Rode, “Trichinella spiralis thymidy- late synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression,”
Parasitology 128 (2004) 209 – 221.
[2] B.K. Shoichet, R.M. Stroud, D.V. Santi, I.D. Kuntz, K.M. Perry, “Structure-based discovery of inhibitors of thymidylate synthase,” Science 259 (1993) 1445 – 1450.
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