Classical Thymidylate Synthase Research Articles

Selected Articles from This Issue

α-FR is overexpressed in a variety of solid tumors, including high-grade serous ovarian cancer, triple-negative breast cancer, and non–small cell lung cancer. CT900 is a small molecule thymidylate synthase (TS) inhibitor that binds to and is taken up by α-FR. Banerjee and colleagues report results from the first clinical trial to show reproducible single agent responses caused by a small molecule in α-FR-overexpressing cancers. The toxicity profile did not show classical TS inhibitor toxicity such as myelosuppression suggesting differential α-FR mediated uptake in tumor tissue. This trial is proof of concept for developing α-FR targeting small molecules, which are selectively taken up in α-FR-overexpressing tumors for patient benefit and are an alternate approach to antibody drug conjugates that already target α-FR.Immune checkpoint inhibitors (ICI) have modest efficacy in patients with mucosal melanoma. Although PD-1/PD-L1 monotherapy has limited benefit, combined therapy with CTLA4 and PD-1 inhibitors improves response rates but increases toxicity. VEGF is highly expressed in melanoma and contributes to progression. Combined treatment with ICI and VEGF inhibitors has shown positive outcomes across various cancer types. Here, Mao and colleagues present the results of a phase II study evaluating the PD-L1 inhibitor atezolizumab and the VEGF inhibitor bevacizumab as first-line therapy in Chinese patients with advanced mucosal melanoma. Over a median follow-up of 13.4 months, the objective response rate was 45.0% in 40 evaluable patients. Atezolizumab in combination with bevacizumab showed promising efficacy and manageable safety. This study sheds light on the dual strategy of VEGF and PD-L1 inhibition in patients with this rare melanoma subtype, for whom prognosis is otherwise generally poor.Defining the immune mechanisms responsible for early control of tumors is critical for guiding treatment strategies. Ferguson and colleagues applied imaging mass cytometry to map the immune landscape and identify differences in immune populations and their specific interactions between high-risk primary cSCC tumors that did not progress and those that developed metastases. The study also reveals that the immunosuppressive state alters the cellular composition of primary tumors. The ability to predict metastatic tumor progression at the time of initial diagnosis of primary cSCC will aid personalized medical care and better disease surveillance.Using publicly available genome-wide genetic dependency maps, Grubb and colleagues sought lineage-enriched anticancer targets. In kidney cancer, this strategy identified BCL2L1 which encodes the antiapoptotic BCL-XL protein as a candidate. Relying on cell-based apoptosis assays, in vivo studies, and clinical validations, as well as using genetic and pharmacological approaches, these studies revealed that a tumor's mesenchymal cell state conferred BCL-XL dependence. The findings identify BCL-XL as a novel target in kidney cancer and present potential biomarkers to stratify renal tumors based on their reliance on BCL-XL.

Study of Kinetic Mechanism of Flavin‐Dependent Thymidylate Synthase from Thermotoga Maritima

Thymidylate, an essential DNA nucleotide, is synthesized de novo by all organisms. The enzyme thymidylate synthase catalyses the last committed step of this de novo pathway, which is the reductive methylation of 2′‐deoxyuridine‐5′‐monophosphate (dUMP) to produce 2′‐deoxythymidine‐5′‐monophosphate (dTMP). Most organism including humans, rely on classical thymidylate synthase. Recently the identification of many bacteria and human pathogens lacking this enzyme led to the discovery of flavin‐dependent thymidylate synthase (FDTS) encoded by the thyX gene. The two classes of enzyme share no structural or sequence homology and while the mechanism of classical enzyme has been studied over decades and is well established, the mechanism of thyX is still not clear. Overall, thyX receives reducing equivalents from NADPH and uses its flavin prosthetic group for redox chemistry and methylenetetrahydrofolate (CH2H4folate) as the methylene donor to produce dTMP with the concomitant release of tetrahydrofolate. Many different mechanisms have been proposed for the oxidative half‐reaction of this enzyme. One of the major questions involving its catalytic mechanisms is the activation of the pyrimidine substrate. Here we tested the feasibility of a recently published mechanism by synthesizing one of the proposed intermediates. We also present kinetic and spectroscopic data that provides more details into the kinetic mechanism of this flavoenzyme.

Alternative Thymidylate Synthase: New Intermediates Suggest New Flavin Mechanism

Flavin-dependent, thymidylate synthase (FDTS, ThyX) catalyzes the last crucial step in the de novo biosynthesis of thymidylate, a DNA building block. Because FDTS is absent in humans while present in several human pathogens, its inhibition is of interest from pharmaceutical standpoint. Unlike classical thymidylate synthase (TSase, ThyA), FDTS bypasses the need for an active-site nucleophile.1 FDTS still has to activate the substrate for the reaction; however, the nucleophile is not an enzymatic residue as in TSase.2 Further understanding of the chemical mechanism of FDTS would greatly facilitate rational design of mechanism-based inhibitors as leads for new class of antibiotic drugs. We will present the trapping of several intermediates3 that shad new light on possible mechanism for this enzyme. By using acid and base as a quenchers in a rapid-quench experiment, we find that the first intermediate yields different trapped compounds in different quesnchers. The second intermediate, on the other hand, forms dTMP product upon base quenching, which is surprising given that the flavin cofactor is still reduced at this stage (according to flavin's UV-vis absorbance spectrum). The recent identification of the base-trapped intermediate and a crystal structure suggest a new path for the catalyzed methylene transfer from the co-factor (methylene tetrahydrofolate) and the substrate dUMP. These new findings may point to a methylene transfer from its folate-donor to the target nucleotide through N5 of the FAD cofactor, in accordance with findings from X-ray crystallography.4 Funding NIH RO1 GM110775

Synthesis and Evaluation of 6‐Aza‐2′‐deoxyuridine Monophosphate Analogs as Inhibitors of Thymidylate Synthases, and as Substrates or Inhibitors of Thymidine Monophosphate Kinase in Mycobacterium tuberculosis

A series of 5-substituted analogs of 6-aza-2'-deoxyuridine 5'-monophosphate, 6-aza-dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin-dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N-atom in 6-aza-dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 μM). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3-(octanamido)prop-1-ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 μM). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N-atom furnished 6-aza-dTMP (1b), which still was recognized as a substrate by TMPKmt.

Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1

Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and K(m) values of the two substrates dUMP and CH(2) H(4) folate were determined to be 15.3 ± 1.25 μM and 0.35 ± 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti-H. pylori inhibitor discovery.

A phase I and pharmacokinetic study of nolatrexed dihydrochloride, a potent thymidylate synthase (TS) inhibitor, combined with docetaxel in patients with advanced cancer

2115 Background: Thymitaq (Th) is a potent and direct inhibitor of TS which has shown activity in patients with head and neck tumors or hepatocarcinoma, as well as in breast, ovarian, and lung cancer models. Because of its lipophilicity and absence of a glutamate moiety, it is not susceptible to certain resistance mechanisms implicated in classical antifolate TS inhibitors, including impaired transport or decreased polyglutamylation. In addition of accumulative evidence of synergism between taxanes and antifolates, Th and docetaxel (Doce) have different mechanisms of action and activity spectrum, as well as nonoverlapping toxicities, which provide a rationale for their evaluation in combination. Methods: Escalating doses of 120-hour IV infusion of Th from a starting dose level of 600 mg/m2/day, and Doce from an initial dose of 30 mg/m2 IV on D1 every 28 days were administered to minimally-pretreated patients with advanced solid malignancies. Folic acid and vitamin B12 supplementation was provided during the study. Results: To date, 8 pts (median age 58, [45–72]; 3 male/5 female; hepatocarcinoma [3], gall bladder, esophagus, stomach, and kidney carcinoma, and soft tissue sarcoma [1, each]) have received 13 courses (range 1–4) at Doce dose levels of 30 mg/m2 (3 pts), and 45 mg/m2 (5 pts) with 600 mg/m2/day of Th. DLTs have been G3 fatigue (1 pt), and G3 mucositis and G4 neutropenia, febrile, in a patient with cirrhosis and hepatocarcinoma, which might have resulted in decreased clearance of the drugs, especially Doce. Toxicities are mostly mild to moderate, including G3 neutropenia, G2 thrombocytopenia, and G2 nausea/vomiting, diarrhea and bilirubin elevation, each in 12.5% of pts. Css of Th (mean, 5.51 μg/mL; ranges 2.86 to 6.19 μg/mL; n=5 pts), were in the same range as prior PK studies at a similar dose and, at the present dose level, already portend biologic activity based on animal models. Conclusions: The MTD and recommended dose of Th in combination with Doce for disease-directed studies with this regimen has not been determined yet. Expanded accrual continues at 600 mg/m2/day of Th and 45 mg/m2 of Doce as the potential recommended dose. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Eximias

Catalytic Mechanism of Chlamydia trachomatis Flavin-dependent Thymidylate Synthase

Here we report on a Chlamydia trachomatis gene that complements the growth defect of a thymidylate synthase-deficient strain of Escherichia coli. The complementing gene encodes a 60.9-kDa protein that shows low level primary sequence homology to a new class of thymidylate-synthesizing enzymes, termed flavin-dependent thymidylate synthases (FDTS). Purified recombinant chlamydial FDTS (CTThyX) contains bound flavin. Results with site-directed mutants indicate that highly conserved arginine residues are required for flavin binding. Kinetic characterization indicates that CTThyX is active as a tetramer with NADPH, methylenetetrahydrofolate, and dUMP required as substrates, serving as source of reducing equivalents, methyl donor, and methyl acceptor, respectively. dTMP and H(4)folate are products of the reaction. Production of H(4)folate rather than H(2)folate, as in the classical thymidylate synthase reaction, eliminates the need for dihydrofolate reductase, explaining the trimethoprim-resistant phenotype displayed by thyA(-) E. coli-expressing CTThyX. In contrast to the extensively characterized thyA-encoded thymidylate synthases, which form a ternary complex with substrates dUMP and CH(2)H(4)folate and follow an ordered sequential mechanism, CTThyX follows a ping-pong kinetic mechanism involving a methyl enzyme intermediate. Mass spectrometry was used to localize the methyl group to a highly conserved arginine, and site-directed mutagenesis showed this arginine to be critical for thymidylate synthesizing activity. These differentiating characteristics clearly distinguish FDTS from ThyA, making this class of enzymes attractive targets for rational drug design.

Mechanistic studies of a flavin-dependent thymidylate synthase.

The ThyA gene that encodes for thymidylate synthase (TS) is absent in the genomes of a large number of bacteria, including several human pathogens. Many of these bacteria also lack the genes for dihydrofolate reductase (DHFR) and thymidine kinase and are totally dependent on an alternative enzyme for thymidylate synthesis. Thy1 encodes flavin-dependent TS (FDTS, previously denoted as TSCP) and shares no sequence homology with classical TS genes. Mechanistic studies of a FDTS from Thermotoga maritima (TM0449) are presented here. Several isotopic labeling experiments reveal details of the catalyzed reaction, and a chemical mechanism that is consistent with the experimental data is proposed. The reaction proceeds via a ping-pong mechanism where nicotinamide binding and release precedes the oxidative half-reaction. The enzyme is primarily pro-R specific with regard to the nicotinamide (NADPH), the oxidation of which is the rate-limiting step of the whole catalytic cascade. An enzyme-bound flavin is reduced with an isotope effect of 25 (consistent with H-tunneling) and exchanges protons with the solvent prior to the reduction of an intermediate methylene. A quantitative assay was developed, and the kinetic parameters were measured. A significant NADPH substrate inhibition and large K(M) rationalized the slow activity reported for this enzyme in the past. These and other findings are compared with classical TS (ThyA) catalysis in terms of kinetic and molecular mechanisms. The differences between the FDTS proposed mechanism and that of the classical TS are striking and invoke the notion that mechanism-based drugs will selectively inhibit FDTS and will not have much effect on human (and other eukaryotes) TS. Since TS activity is essential to DNA replication, the unique mechanism of FDTS makes it an attractive target for antibiotic drug development.

2‐amino‐4‐oxo‐6‐substituted‐pyrrolo[2,3‐d]pynmidines as potential inhibitors of thymidylate synthase

AbstractClassical, antifolate inhibitors of thymidylate synthase often suffer from a number of potential disadvantages when used as antitumor agents. These include impaired uptake due to an alteration of the active transport system required for cellular uptake, as well as the formation of long acting, non‐effluxing polygluta‐mates via folypolyglutamate synthetase, which are responsible for toxicity to normal cells. To overcome some of the disadvantages of classical thymidylate synthase inhibitors, there has been considerable interest in the synthesis and evaluation of nonclassical inhibitors, which could enter cells via passive diffusion and are not substrates for folypolyglutamate synthetase. A series of eight nonclassical 6‐substituted 2‐amino‐4‐oxo‐pyrrolo[2,3‐d]pyrimidines 2a‐2h were designed as potential inhibitors of thymidylate synthase. The synthesis of the target compounds 2a‐2h was achieved via regioselective iodination at the 6‐position of 5, palladium‐catalyzed coupling with the appropriate phenylacetylenes, reduction of the C8‐C9 triple bond followed by saponification. Preliminary biological results indicated that none of the target compounds showed inhibitory activities against thymidylate synthase from Escherichia coli, Lactobacillus casei, rat or human thymidylate synthase at the concentrations tested. None of the target compounds showed inhibitory activity against dihydrofolate reductase from Escherichia coli, Lactobacillus casei, rat or human at 3.0 × 10−5 M. However, 50% inhibition of dihydrofolate reductase from Pneumocystis carinii and from Toxoplasma gondii was achieved with compound 2d and with compound 2g at 3.0 × 10−5 M.

Thymidylate synthase inhibition: a structure-based rationale for drug design.

Thymidylate synthase (TS) is a very interesting target in antiproliferative diseases. Its inhibition causes thimineless death of the cells and compounds inhibiting TS are widely used in anticancer therapy. The classical antifolate TS inhibitors are structural analogs of the folate cofactor; they often share the same metabolic pathways and this causes the development of resistance inside the cells. A detailed analysis of the available x-ray crystal structures of the complexes of the enzyme with different substrates and inhibitors support the finding of a structural basis of their biological activity. TS inhibitors nonstructural analog of folate, non-analog antifolate inhibitors (NAAI), are welcome as a new interesting research topic. Among the most recent and interesting ones, compounds from Agouron related to the indole structure, are independent on the folate metabolism, highly active and specific for human TS. Other compounds, phthalein derivatives, can inhibit TS enzymes from various sources and show an interesting biological activity profile: they inhibit better bacterial and fungal TS than human TS. The x-ray crystal structures of some of these inhibitors with TS show that they bind in a different binding site from that of the classical folate TS inhibitors. This indicates a potential allosteric binding site useful for future drug discovery studies.