Multi-targeted antifolates aimed at avoiding drug resistance form covalent closed inhibitory complexes with human and Escherichia coli thymidylate synthases

Sayre, Peter H.; Finer-Moore, J.; Fritz, Timothy A.; Biermann, D.; Gates, Susan B.; MacKellar, W.C.; Patel, Vinod F.; Stroud, Robert M.

doi:10.1006/jmbi.2001.5074

Cited by 58 publications

(54 citation statements)

References 42 publications

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“…Some of the 74 different replacements (or combinations thereof) are responsible for 5-FdUR resistance, including the substitutions in single mutants, whereas other replacements are presumably co-selected and not relevant to resistance. Recent crystallographic analyses of TS, especially closed ternary complexes with dUMP and folate-based inhibitors (14,16), allow us to evaluate these replacements with a view toward understanding possible structure-function relationships and establishing fresh targets for directed mutagenesis. We have used the atomic coordinates of the wild-type amino acids in tightly closed complexes with dUMP and a folate analog inhibitor (see Refs.…”

Section: Discussionmentioning

confidence: 99%

“…Possible resistance mechanisms can be envisioned for some of the replacements, whereas no mechanisms are apparent for others. For example, substitution of valine for glutamate in the D48V mutant would disrupt the hydrogen bonding network within the Arg 50 loop that mediates dUMP binding and also affect interactions with the second subunit of the obligate homodimer (14,16); it has been proposed that mutations at residues 47-52 may confer resistance by destabilizing the closed ternary complex (16). At position 84 in the V84A mutant, wild-type valine interacts with two residues, Phe 80 and Phe 225 , that contact the cofactor.…”

Section: Discussionmentioning

confidence: 99%

“…This region has not been reported to be involved in 5-FdUR resistance; structural analysis indicates that it is located on the active site pocket and interacts with folate-based inhibitors, providing a plausible rationale for resistance (1, 11-16). The N-terminal residues from Gly 5 through Gln 18 accumulated 10 substitutions; this segment is disordered in existing crystal structures and has no known function (11)(12)(13)(14)(15)(16). There is no obvious correlation between the frequency of either single or multiple substitutions observed in resistant mutants and the presence of ␤-sheets or ␣-helices.…”

Section: Distribution Of Pcr-generated Mutations Yielding 5-fdurmentioning

confidence: 99%

“…Among these site-directed replacements is T51S, the most common mutation we have previously observed and present in the highly resistant variant T51S, G52S (6). Thr 51 participates in hydrogen bonding within the Arg 50 loop and with Val 313 after ligand-induced conformational changes of the C-terminal segment (1,(11)(12)(13)(14)(15)(16); 5-FdUR resistance could arise at Thr 51 from several mechanisms, including an effect on this conformational change. Two replacements at Asp 254 (D254N and D254E) were observed in multiply mutated variants (255, 318, 358), and we found that both, as well as the D254A substitution, conferred resistance as single mutations (Fig.…”

Section: -Fdur-resistant Thymidylate Synthase Mutantsmentioning

confidence: 99%

“…Although metabolically unstable, the P303L protein was nonetheless able to confer resistance against both 5-FdUR-and anti-folate-based TS inhibitors in transfected cells (10). Like Y33H, the P303L substitution is not in close proximity to the catalytic site as determined from crystal structures (11)(12)(13)(14)(15)(16).…”

mentioning

confidence: 99%

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Distribution of Mutations in Human Thymidylate Synthase Yielding Resistance to 5-Fluorodeoxyuridine

Kawate¹,

Landis²,

Loeb³

2002

Journal of Biological Chemistry

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Thymidylate synthase (TS) catalyzes methylation of dUMP to dTMP and is the target of cancer chemotherapeutic agents (e.g. 5-fluorouracil). Here, we used errorprone PCR to mutagenize the full-length human TS cDNA and then selected mutants resistant to 5-fluorodeoxyuridine in a bacterial complementation system. We found that resistant mutants contained 1-5 amino acid substitutions and that these substitutions were located along the entire length of the polypeptide. Many of these residues are distant from the active site and/or have no documented function in catalysis or resistance. We conclude that mutations distributed throughout the linear sequence and threedimensional structure of human TS can confer resistance to 5-fluorodeoxyuridine. Our findings imply that long range interactions within proteins affect catalysis at the active site and that mutations at a distance can yield variant proteins with desired properties.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Distribution Of Pcr-generated Mutations Yielding 5-fdurmentioning

confidence: 99%

Section: -Fdur-resistant Thymidylate Synthase Mutantsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Distribution of Mutations in Human Thymidylate Synthase Yielding Resistance to 5-Fluorodeoxyuridine

Kawate¹,

Landis²,

Loeb³

2002

Journal of Biological Chemistry

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Understanding the structural basis of species selective, stereospecific inhibition for Cryptosporidium and human thymidylate synthase

et al. 2019

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Thymidylate synthase (TS), found in all organisms, is an essential enzyme responsible for the de novo synthesis of deoxythymidine monophosphate. The TS active sites of the protozoal parasite Cryptosporidium hominis and human are relatively conserved. Evaluation of antifolate compound 1 and its R‐enantiomer 2 against both enzymes reveals divergent inhibitor selectivity and enzyme stereospecificity. To establish how C. hominis and human TS (ChTS and hTS) selectively discriminate 1 and 2, respectively, we determined crystal structures of ChTS complexed with 2 and hTS complexed with 1 or 2. Coupled with the previously determined structure of ChTS complexed with 1, we discuss a possible mechanism for enzyme stereospecificity and inhibitor selectivity.

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Segmental motions of rat thymidylate synthase leading to half‐the‐sites behavior

et al. 2010

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Thymidylate synthase (TS) is a homodimeric enzyme with two equivalent active sites composed of residues from both subunits. Despite the structural symmetry of the enzyme, certain experimental results are consistent with half-the-sites activity, suggesting negative cooperativity between the active sites. To gain insight into the mechanism behind this phenomenon, we explore segmental motions of rat TS in the absence of ligands, with normal mode analysis as a tool. Using solvent accessible surface area of the active site pocket as a monitor of the degree of opening of the active sites, we classified the first 25 nontrivial normal modes, obtained from the web server of the program ElNémo, according to the behavior of the active sites. We found seven modes that open and close both sites symmetrically and nine that do so in an anticorrelated fashion. We characterized the motions of these modes by visual inspection and through measurement of distances between selected atoms lining the active site pockets. The segments that regulate access to the active site correspond to the loop containing R44, helix K, and a long loop containing residues 103-125, in agreement with a large body of crystallographic studies. These elements can be activated together or in isolation. There are more asymmetric modes than symmetric ones in the set we analyzed, probably accounting for the half-the-sites behavior of the enzyme. Three of the asymmetric modes result in changes at the dimer interface and indicate the endpoints of possible communication pathways between the active sites.

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Multi-targeted antifolates aimed at avoiding drug resistance form covalent closed inhibitory complexes with human and Escherichia coli thymidylate synthases

Cited by 58 publications

References 42 publications

Distribution of Mutations in Human Thymidylate Synthase Yielding Resistance to 5-Fluorodeoxyuridine

Distribution of Mutations in Human Thymidylate Synthase Yielding Resistance to 5-Fluorodeoxyuridine

Understanding the structural basis of species selective, stereospecific inhibition for Cryptosporidium and human thymidylate synthase

Segmental motions of rat thymidylate synthase leading to half‐the‐sites behavior

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