Human 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 : mechanism of 2'-deoxyuridine 5'-monophosphate hydrolysis

Funding: Industrial Biotechnology Innovation Centre - 2021-01-01.The enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase 1 (DNPH1) catalyzes the N-ribosidic bond cleavage of 5-hydroxymethyl-2′-deoxyuridine 5′-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2′-deoxynucleoside 5′-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2′-deoxyuridine 5′-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography–mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid–base catalysis is operational and that for maximum kcat/KM, two groups with an average pKa of 6.4 must be deprotonated, while two groups with an average pKa of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to kcat/KM. Solvent deuterium isotope effects on kcat/KM and kcat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.Publisher PDFPeer reviewe

Snapshots of the reaction coordinate of a thermophilic 2'-deoxyribonucleoside/ribonucleoside transferase

Funding: P.T. is funded by IBioIC (IBioIC 2020-2-1), and C.M.C. is funded by the Wellcome Trust (217078/Z/19/Z). C.M.C. and D.H. are funded by research grants from NuCana plc..Nucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2′-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2′-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2′-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2′-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis (CtNDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5–9.5, with two very distinct pKa values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2′-fluoro-2′-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2′-difluoro-2′-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2′-deoxy)ribonucleoside selection, as CtNDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of CtNDT in biocatalysis.Peer reviewe

Pre- and post-selected ensembles and time-symmetry in quantum mechanics

An expression is proposed for the quantum mechanical state of a pre- and post-selected ensemble, which is an ensemble determined by the final as well as the initial state of the quantum systems involved. It is shown that the probabilities calculated from the proposed state agree with previous expressions, for cases where they both apply. The same probabilities are found when they are calculated in the forward- or reverse-time directions. This work was prompted by several problems raised by Shimony recently in relation to the state, and time symmetry, of pre- and post-selected ensembles.Comment: RevTex4, 17 pages, no fig

Phase II randomised discontinuation trial of brivanib in patients with advanced solid tumours

Background: Brivanib is a selective inhibitor of vascular endothelial growth factor and fibroblast growth factor (FGF) signalling. We performed a phase II randomised discontinuation trial of brivanib in 7 tumour types (soft-tissue sarcomas [STS], ovarian cancer, breast cancer, pancreatic cancer, non-small-cell lung cancer [NSCLC], gastric/esophageal cancer and transitional cell carcinoma [TCC]). Patients and methods: During a 12-week open-label lead-in period, patients received brivanib 800 mg daily and were evaluated for FGF2 status by immunohistochemistry. Patients with stable disease at week 12 were randomised to brivanib or placebo. A study steering committee evaluated week 12 response to determine if enrolment in a tumour type would continue. The primary objective was progression-free survival (PFS) for brivanib versus placebo in patients with FGF2-positive tumours. Results: A total of 595 patients were treated, and stable disease was observed at the week 12 randomisation point in all tumour types. Closure decisions were made for breast cancer, pancreatic cancer, NSCLC, gastric cancer and TCC. Criteria for expansion were met for STS and ovarian cancer. In 53 randomised patients with STS and FGF2-positive tumours, the median PFS was 2.8 months for brivanib and 1.4 months for placebo (hazard ratio [HR]: 0.58, p = 0.08). For all randomised patients with sarcomas, the median PFS was 2.8 months (95% confidence interval [CI]: 1.4–4.0) for those treated with brivanib compared with 1.4 months (95% CI: 1.3–1.6) for placebo (HR = 0.64, 95% CI: 0.38–1.07; p = 0.09). In the 36 randomised patients with ovarian cancer and FGF2-positive tumours, the median PFS was 4.0 (95% CI: 2.6–4.2) months for brivanib and 2.0 months (95% CI: 1.2–2.7) for placebo (HR: 0.56, 95% CI: 0.26–1.22). For all randomised patients with ovarian cancer, the median PFS in those randomised to brivanib was 4.0 months (95% CI: 2.6–4.2) and was 2.0 months (95% CI: 1.2–2.7) in those randomised to placebo (HR = 0.54, 95% CI: 0.25–1.17; p = 0.11). Conclusion: Brivanib demonstrated activity in STS and ovarian cancer with an acceptable safety profile. FGF2 expression, as defined in the protocol, is not a predictive biomarker of the efficacy of brivanib