Alanine mutants of the interface residues of human thymidylate synthase decode key features of the binding mode of allosteric anticancer peptides.
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ABSTRACT:
SUBMITTER: Tochowicz A
PROVIDER: S-EPMC8346289 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The parasite Toxoplasma gondii can lead to toxoplasmosis in those who are immunocompromised. To combat the infection, the enzyme responsible for nucleotide synthesis thymidylate synthase-dihydrofolate reductase (TS-DHFR) is a suitable drug target. We have used virtual screening to determine novel allosteric inhibitors at the interface between the two TS domains. Selected compounds from virtual screening inhibited TS activity. Thus, these results show that allosteric inhibition by small drug-like molecules can occur in T. gondii TS-DHFR and pave the way for new and potent species-specific inhibitors.
| S-EPMC3946055 | biostudies-literature
Project description:Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.
| S-EPMC3161595 | biostudies-literature
Project description:Thymidylate synthase (TS) is a prominent drug target for different cancer types. However, the prolonged use of its classical inhibitors, substrate analogs that bind at the active site, leads to TS overexpression and drug resistance in the clinic. In the effort to identify anti-TS drugs with new modes of action and able to overcome platinum drug resistance in ovarian cancer, octapeptides with a new allosteric inhibition mechanism were identified as cancer cell growth inhibitors that do not cause TS overexpression. To improve the biological properties, 10 cyclic peptides (cPs) were designed from the lead peptides and synthesized. The cPs were screened for the ability to inhibit recombinant human thymidylate synthase (hTS), and peptide 7 was found to act as an allosteric inhibitor more potent than its parent open-chain peptide [Pro3]LR. In cytotoxicity studies on three human ovarian cancer cell lines, IGROV-1, A2780, and A2780/CP, peptide 5 and two other cPs, including 7, showed IC50 values comparable with those of the reference drug 5-fluorouracil, of the open-chain peptide [d-Gln4]LR, and of another seven prolyl derivatives of the lead peptide LR. These promising results indicate cP 7 as a possible lead compound to be chemically modified with the aim of improving both allosteric TS inhibitory activity and anticancer effectiveness.
| S-EPMC6804113 | biostudies-literature
Project description:Thymidylate synthase (TS) is a target in the chemotherapy of colorectal cancer and some other neoplasms. It catalyzes the transfer of a methyl group from methylenetetrahydrofolate to dUMP to form dTMP. On the basis of structural considerations, we have introduced 1,3-propanediphosphonic acid (PDPA) as an allosteric inhibitor of human TS (hTS); it is proposed that PDPA acts by stabilizing an inactive conformer of loop 181-197. Kinetic studies showed that PDPA is a mixed (noncompetitive) inhibitor versus dUMP. In contrast, versus methylenetrahydrofolate at concentrations lower than 0.25 microM, PDPA is an uncompetitive inhibitor, while at PDPA concentrations higher than 1 microM the inhibiton is noncompetive, as expected. At the concentrations corresponding to uncompetitive inhibition, PDPA shows positive cooperativity with an antifolate inhibitor, ZD9331, which binds to the active conformer. PDPA binding leads to the formation of hTS tetramers, but not higher oligomers. These data are consistent with a model in which hTS exists preferably as an asymmetric dimer with one subunit in the active conformation of loop 181-197 and the other in the inactive conformation.
| S-EPMC2516748 | biostudies-literature
Project description:Drug-target interaction, cellular internalization, and target engagement should be addressed to design a lead with high chances of success in further optimization stages. Accordingly, we have designed conjugates of folic acid with anticancer peptides able to bind human thymidylate synthase (hTS) and enter cancer cells through folate receptor α (FRα) highly expressed by several cancer cells. Mechanistic analyses and molecular modeling simulations have shown that these conjugates bind the hTS monomer-monomer interface with affinities over 20 times larger than the enzyme active site. When tested on several cancer cell models, these conjugates exhibited FRα selectivity at nanomolar concentrations. A similar selectivity was observed when the conjugates were delivered in synergistic or additive combinations with anticancer agents. At variance with 5-fluorouracil and other anticancer drugs that target the hTS catalytic pocket, these conjugates do not induce overexpression of this protein and can thus help combating drug resistance associated with high hTS levels.
| S-EPMC8041318 | biostudies-literature
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a highly infectious human herpesvirus that causes Kaposi's sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2'-deoxyuridine-5'-monophosphate (dUMP) to thymidine-5'-monophosphate (dTMP) using 5,10-methylenetetrahydrofolate (mTHF) as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo), complexes with dUMP (binary), and complexes with both dUMP and raltitrexed (ternary) were determined at 1.7 Å, 2.0 Å, and 2.4 Å, respectively. While the ternary complex structures of human thymidylate synthase and E. coli thymidylate synthase had a closed conformation, the ternary complex structure of KSHV thymidylate synthase was observed in an open conformation, similar to that of rat thymidylate synthase. The complex structures of KSHV thymidylate synthase did not have a covalent bond between the sulfhydryl group of Cys219 and C6 atom of dUMP, unlike the human thymidylate synthase. The catalytic Cys residue demonstrated a dual conformation in the apo structure, and its sulfhydryl group was oriented toward the C6 atom of dUMP with no covalent bond upon ligand binding in the complex structures. These structural data provide the potential use of antifolates such as raltitrexed as a viral induced anticancer drug and structural basis to design drugs for targeting the thymidylate synthase of KSHV.
| S-EPMC5148040 | biostudies-literature
Project description:In human cells, thymidylate synthase (TS) provides the only source of 2'-deoxythymidyne-5'-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking the hTS active site has limitations due to the onset of resistance in cancer cells, requiring the identification of new strategies to effectively inhibit this enzyme. Human TS works as an obligate homodimer, making the inter-subunit interface an attractive targetable area. Here, we report the design and investigation of a new hTS variant, in which Gln62, located at the dimer interface, has been replaced by arginine in order to destabilize the enzyme quaternary assembly. The hTS Q62R variant has been characterized though kinetic assay, thermal denaturation analysis and X-ray crystallography. Our results provide evidence that hTS Q62R has a reduced melting temperature. The effective destabilization of the TS quaternary structure is also confirmed by structural analysis, showing that the introduced mutation induces a slight aperture of the hTS dimer. The generation of hTS variants having a more accessible interface area can facilitate the screening of interface-targeting molecules, providing key information for the rational design of innovative hTS interface inhibitors.
| S-EPMC6523895 | biostudies-literature
Project description:BackgroundThymidylate synthase is a housekeeping gene, designated ancient due to its role in DNA synthesis and ubiquitous phyletic distribution. The genomic sequences were characterized coding for thymidylate synthase in two species of the genus Trichinella, an encapsulating T. spiralis and a non-encapsulating T. pseudospiralis.MethodsBased on the sequence of parasitic nematode Trichinella spiralis thymidylate synthase cDNA, PCR techniques were employed.ResultsEach of the respective gene structures encompassed 6 exons and 5 introns located in conserved sites. Comparison with the corresponding gene structures of other eukaryotic species revealed lack of common introns that would be shared among selected fungi, nematodes, mammals and plants. The two deduced amino acid sequences were 96% identical. In addition to the thymidylate synthase gene, the intron-less retrocopy, i.e. a processed pseudogene, with sequence identical to the T. spiralis gene coding region, was found to be present within the T. pseudospiralis genome. This pseudogene, instead of the gene, was confirmed by RT-PCR to be expressed in the parasite muscle larvae.ConclusionsIntron load, as well as distribution of exon and intron phases in thymidylate synthase genes from various sources, point against the theory of gene assembly by the primordial exon shuffling and support the theory of evolutionary late intron insertion into spliceosomal genes. Thymidylate synthase pseudogene expressed in T. pseudospiralis muscle larvae is designated a retrogene.
| S-EPMC4022200 | biostudies-literature
Project description:The allosteric enzyme, l-lactate dehydrogenase (LDH), is activated by fructose 1,6-metaphosphate (FBP) to reduce pyruvate to lactate. The molecular details of the FBP-driven transition from the low affinity T state to the high affinity R state in LDH, a tetramer composed of identical subunits, are not known. The dynamics of the T → R allosteric transition, investigated using Brownian dynamics (BD) simulations of the self-organized polymer (SOP) model, revealed that coordinated rotations of the subunits drive the T → R transition. We used the structural perturbation method (SPM), which requires only the static structure, to identify the allostery wiring diagram (AWD), a network of residues that transmits signals across the tetramer, as LDH undergoes the T → R transition. Interestingly, the residues that play a major role in the dynamics, which are predominantly localized at the interfaces, coincide with the AWD identified using the SPM. Although the allosteric pathways are highly heterogeneous, on the basis of our simulations, we surmise that predominantly the conformational changes in the T → R transition start from the region near the active site, comprised of helix αC, helix α1/2G, helix α3G, and helix α2F, and proceed to other structural units, thus completing the global motion. Brownian dynamics simulations of the tetramer assembly, triggered by a temperature quench from the fully disrupted conformations, show that the bottleneck for assembly is the formation of the correct orientational registry between the subunits, requiring contacts between the interface residues. Surprisingly, these residues are part of the AWD, which was identified using the SPM. Taken together, our results show that LDH, and perhaps other multidomain proteins, may have evolved to stabilize distinct states of allosteric enzymes using precisely the same AWD that also controls the functionally relevant allosteric transitions.
| S-EPMC6422748 | biostudies-literature
Project description: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.
| S-EPMC4634673 | biostudies-literature