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Hotspots in an obligate homodimeric anticancer target. Structural and functional effects of interfacial mutations in human thymidylate synthase.

ABSTRACT: Human thymidylate synthase (hTS), a target for antiproliferative drugs, is an obligate homodimer. Single-point mutations to alanine at the monomer-monomer interface may enable the identification of specific residues that delineate sites for drugs aimed at perturbing the protein-protein interactions critical for activity. We computationally identified putative hotspot residues at the interface and designed mutants to perturb the intersubunit interaction. Dimer dissociation constants measured by a FRET-based assay range from 60 nM for wild-type hTS up to about 1 mM for single-point mutants and agree with computational predictions of the effects of these mutations. Mutations that are remote from the active site retain full or partial activity, although the substrate KM values were generally higher and the dimer was less stable. The lower dimer stability of the mutants can facilitate access to the dimer interface by small molecules and thereby aid the design of inhibitors that bind at the dimer interface.

SUBMITTER: Salo-Ahen OM

PROVIDER: S-EPMC4634673 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Hotspots in an obligate homodimeric anticancer target. Structural and functional effects of interfacial mutations in human thymidylate synthase.

Salo-Ahen Outi M H OM Tochowicz Anna A Pozzi Cecilia C Cardinale Daniela D Ferrari Stefania S Boum Yap Y Mangani Stefano S Stroud Robert M RM Saxena Puneet P Myllykallio Hannu H Costi Maria Paola MP Ponterini Glauco G Wade Rebecca C RC

Journal of medicinal chemistry 20150401 8

Human thymidylate synthase (hTS), a target for antiproliferative drugs, is an obligate homodimer. Single-point mutations to alanine at the monomer-monomer interface may enable the identification of specific residues that delineate sites for drugs aimed at perturbing the protein-protein interactions critical for activity. We computationally identified putative hotspot residues at the interface and designed mutants to perturb the intersubunit interaction. Dimer dissociation constants measured by a ...[more]

PMID: 25798950

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Project description:Background5-fluorouracil (5-FU) and mitomycin-C (MMC) with radiotherapy (RT) remain an established treatment for patients with anal cancer (AC). Genetic mutations in two major metabolizing enzymes for 5-FU; dihydropyrimidine dehydrogenase (DPYD and thymidylate synthetase (TYMS), have been associated with clinical response and toxicity. However, their place in the treatment of AC remains undetermined.MethodsWe retrospectively reviewed 21 patients with AC, including T2-4, N0-1, M0 or T1-4, N2-3, and M0 treated between 2012 and 2018. All patients were treated with 5-FU 1,000 mg/m2/day via continuous intravenous (IV) infusion 1-4 and 29-32, MMC 10 mg/m2 IV bolus days 1 and 29 plus RT. Patients who developed ⩾3 grade toxicities were tested for the DPYD and TYMS genes. Treatment was either modified with reduced doses or changed to MMC 10 mg/m2 day 1 and 29 with cisplatin 25 mg/m2/week plus RT. Toxicities and responses were collected.ResultsSix out of 21 patients who developed ⩾3 grade toxicities including pancytopenia, neutropenia, thrombocytopenia, mucositis, nausea, rash, and nephritis were found to have genetic mutations: TYMS 2RG/3RC (n = 2), 3RG/3RC (n = 1), 2R/2R (n = 2), TYMS 3'UTR del/Ins (n = 2), and DPYD c.2864A > T heterozygous (n = 1). Two patients received 5-FU at a 50% reduced dose on days 29-32; one patient refused to receive 5-FU (continued with MMC and RT); one patient received only radiation therapy due to persistent pancytopenia despite the use of growth factors; two patients received an alternative regimen consisting of MMC 10 mg/m2 on day 29 with cisplatin (CDDP) 25 mg/m2/week plus RT; and two patients received cisplatin/MMC with RT from the beginning as they were prospectively detected to have TYMS abnormalities prior to dosing the chemotherapy. These patients tolerated treatment very well with only grade 2 toxicities. All the patients (4/4) on cisplatin/MMC achieved clinical complete response (cCR), while four patients (4/15) on 5-FU/MMC reached cCR at the first assessment. Radiological response showed complete response at the end of 24 weeks assessment.ConclusionsMolecular testing for DPYD and TYMS genes can allow us to identify patients who are most likely to respond or face severe toxicity to 5-FU in a potentially curable cancer. Combining radiation with CDDP with MMC in patients with AC is feasible. A prospective study based on pharmacogenetic testing comparing MMC/cisplatin with MMC/5-FU is indicated in patients with AC.

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Hotspots in an obligate homodimeric anticancer target. Structural and functional effects of interfacial mutations in human thymidylate synthase.

Publications

Hotspots in an obligate homodimeric anticancer target. Structural and functional effects of interfacial mutations in human thymidylate synthase.

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