Structure-based studies on species-specific inhibition of thymidylate synthase

Costi, Maria Paola; Tondi, Donatella; Rinaldi, Marcella; Barlocco, Daniela; Pecorari, P.; Soragni, Fabrizia; Venturelli, Alberto; Stroud, Robert M.

doi:10.1016/s0925-4439(02)00083-2

Cited by 41 publications

(43 citation statements)

References 30 publications

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“…Compounds, including MR36 used in the present study ( Fig. 1), have been designed specifically to avoid polyglutamylation and to enter the cell through membrane diffusion [16].…”

Section: Introductionmentioning

confidence: 99%

Biological evaluation of MR36, a novel non-polyglutamatable thymidylate synthase inhibitor that blocks cell cycle progression in melanoma cell lines

et al. 2011

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Melanoma is one of the most common cancers, and its incidence has continued to increase over the past few decades. Chemotherapy resistance and related defects in apoptotic signaling are critical for the high mortality of melanoma. Effective drugs are lacking because apoptosis regulation in this tumor type is not well understood. The folate pathway has been considered an interesting target for anticancer therapies, and approaches targeting this pathway have recently been extended to melanoma treatment. In this study, the intracellular apoptosis signaling pathways of two melanoma cells lines (SK-MEL-2 and SK-MEL-28) were investigated after treatment with a new experimental antifolate substance (MR36) that targets thymidylate synthase. In both melanoma cell lines, apoptosis induction was triggered by a p53-independent mechanism. MR36-induced apoptosis was associated with a loss of both mitochondrial membrane potential and caspase-3 activation. Induction of cell cycle arrest by MR36 was associated with changes in the expression of key cell cycle regulators, such as p21 and cyclin D1, and the hypophosphorylation of pRb. In addition, Fas signaling was also analyzed. These findings suggest that, unlike classical antifolates, MR36 exerted an inhibitory effect on both the enzymatic function and expression of thymidylate synthase, thereby inducing apoptosis through the activation of the extrinsic and intrinsic pathways in the melanoma cell lines. MR36 showed a different mechanism of action from the known antifolates (Nolatrexed and Pemetrexed) that resulted in higher anticancer activity. Therefore, MR36 should be included as a potential new therapeutic treatment in melanoma research.

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“…Compounds, including MR36 used in the present study ( Fig. 1), have been designed specifically to avoid polyglutamylation and to enter the cell through membrane diffusion [16].…”

Section: Introductionmentioning

confidence: 99%

Biological evaluation of MR36, a novel non-polyglutamatable thymidylate synthase inhibitor that blocks cell cycle progression in melanoma cell lines

et al. 2011

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“…This enzyme converts deoxyuridine monophosphate (dUMP) to thymidine monophosphate (dTMP), an activity essential for DNA synthesis. The cancer drug 5-fluorouracil irreversibly inhibits TS, and a selective inhibitor of a non-human form of the enzyme could yield a new antibiotic or antifungal drug [11].…”

Section: Thymidylate Synthase: Proof Of Principlementioning

confidence: 99%

Tethering

Erlanson¹,

Ballinger²,

Wells³

2006

Methods and Principles in Medicinal Chemistry

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IntroductionOne of the common challenges throughout this book is how to identify small chemical fragments that bind weakly to target biological molecules. Among fragment-based approaches, Tethering is unique in using a covalent, reversible bond to stabilize the interaction between a fragment and a target protein [1,2].The general process is outlined in Fig. 14.1. First, a cysteine residue is either coopted or introduced in a target protein. Metaphorically, the cysteine residue serves as a fishing line to capture fragments (fish) that bind near the cysteine. The protein is incubated with pools of thiol-containing, small molecule fragments which are conjugated to a common, hydrophilic thiol (such as cysteamine) for improved water solubility. By controlling the redox conditions in the experiment with exogenous reducing agents, equilibria can be established so that the cysteine residue in the protein reversibly forms disulfide bonds with the individual fragments. In the absence of any affinity between a fragment and the protein, no fragment should bind more favorably than any other; a pool of fragments produces a statistical mixture of different protein-fragment complexes, as well as unmodified protein and cysteaminemodified protein. However, if a fragment has inherent affinity for the protein and binds near the cysteine residue, the fragment-protein conjugate is stabilized, and this complex predominates. A fragment thus selected can be easily identified by mass spectrometry of the equilibrium mixture, and if each fragment in a pool has a unique molecular weight, so do the resulting protein-fragment conjugates. While the identified fragments are often weak ligands, X-ray crystallography of the protein-fragment conjugates is often facilitated by the covalent bond. These captured fragments then serve as starting points for conversion to non-covalent ligands by chemical optimization and removal of the thiol functionality.In the following pages, we present an overview of the theory and uses of Tethering. After first considering the basis of the technique in thermodynamic terms (section 14.2), we show how the technology can be used in the active sites of enzymes to identify fragments (section 14.4), which can then be elaborated to more potent inhibitors. Section 14.5 considers how Tethering can be used to not only 285 Fragment-based Approaches in Drug Discovery. Edited

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“…As a consequence, TS has been a logical and uniquely suited target for the design of chemotherapeutic agents [11]. In search for specific and non-toxic inhibitors of TS, compounds modeled after both dUMP [12][13][14][15][16] and mTHF [17][18][19][20], as well as novel classes of inhibitors [21][22][23][24][25], have been studied, with 5-fluorouracil [26,27] (after metabolic conversion to 5-fluoro-dUMP (FdUMP)) and folate-based raltitrexed (Tomudex, ZD1694) [18] approved for therapies, and many others, especially antifolates, in different stages of clinical trials. Studies leading scientists to the design of these inhibitors, initiated from simple chemical model systems, kinetic measurements, and spectroscopic data, and more recently reinforced by availability of three-dimensional structures from crystallographic experiments and structure-based design, have resulted in a proposed mechanism of reaction for thymidylate synthase, shown in Figure 1.…”

Section: Introductionmentioning

confidence: 99%

5,10-Methylene-5,6,7,8-tetrahydrofolate conformational transitions upon binding to thymidylate synthase: molecular mechanics and continuum solvent studies

Jarmuła

Cieplak

Montfort

2005

J Comput Aided Mol Des

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We applied the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach to evaluate relative stability of the extended (flat) and C-shaped (bent) solution conformational forms of the 5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) molecule in aqueous solution. Calculations indicated that both forms have similar free energies in aqueous solution but detailed energy components are different. The bent solution form has lower intramolecular electrostatic and van der Waals interaction energies. The flat form has more favorable solvation free energy and lower contribution from the bond, angle and torsion angle molecular mechanical internal energies. We exploit these results and combine them with known crystallographic data to provide a model for the progressive binding of the mTHF molecule, a natural cofactor of thymidylate synthase (TS), to the complex forming in the TS-catalyzed reaction. We propose that at the time of initial weak binding in the open enzyme the cofactor molecule remains in a close balance between the flat and bent solution conformations, with neither form clearly favored. Later, thymidylate synthase undergoes conformational change leading to the closure of the active site and the mTHF molecule is withdrawn from the solvent. That effect shifts the thermodynamic equilibrium of the mTHF molecule toward the bent solution form. At the same time, burying the cofactor molecule in the closed active site produces numerous contacts between mTHF and protein that render change in the shape of the mTHF molecule. As a result, the bent solution conformer is converted to more strained L-shaped bent enzyme conformer of the mTHF molecule. The strain in the bent enzyme conformation allows for the tight binding of the cofactor molecule to the productive ternary complex that forms in the closed active site, and facilitates the protonation of the imidazolidine N10 atom, which promotes further reaction.

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Structure-based studies on species-specific inhibition of thymidylate synthase

Cited by 41 publications

References 30 publications

Biological evaluation of MR36, a novel non-polyglutamatable thymidylate synthase inhibitor that blocks cell cycle progression in melanoma cell lines

Biological evaluation of MR36, a novel non-polyglutamatable thymidylate synthase inhibitor that blocks cell cycle progression in melanoma cell lines

Tethering

5,10-Methylene-5,6,7,8-tetrahydrofolate conformational transitions upon binding to thymidylate synthase: molecular mechanics and continuum solvent studies

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