A fragment-based approach to probing adenosine recognition sites by using dynamic combinatorial chemistry

Abstract

A new strategy that combines the concepts of fragment-based drug design and dynamic combinatorial chemistry (DCC) for targeting adenosine recognition sites on enzymes is reported. We demonstrate the use of 5'-deoxy-5'-thioadenosine as a noncovalent anchor fragment in dynamic combinatorial libraries templated by Mycobacterium tuberculosis pantothenate synthetase. A benzyl disulfide derivative was identified upon library analysis by HPLC. Structural and binding studies of protein-ligand complexes by X-ray crystallography and isothermal titration calorimetry informed the subsequent optimisation of the DCC hit into a disulfide containing the novel meta-nitrobenzyl fragment that targets the pantoate binding site of pantothenate synthetase. Given the prevalence of adenosine-recognition motifs in enzymes, our results provide a proof-of-concept for using this strategy to probe adjacent pockets for a range of adenosine binding enzymes, including other related adenylate-forming ligases, kinases, and ATPases, as well as NAD(P)(H), CoA and FAD(H2) binding proteins.

Figure 1

Biophysical characterisation of 5′-deoxy-5′-thioadenosine (6) binding to M. tuberculosis pantothenate synthetase. A) ¹H NMR spectrum of 6, observing the purine H-2 and H-8 signals. WaterLOGSY experiments B) with no protein, C) with 12 μm pantothenate synthetase, and D) displacement with 330 μm ATP. E) ITC titration of 6. Data were fitted to a single-binding-site model. F) Crystal structure of 6 bound in the enzyme active site. Omit electron density F_o–F_c around 6 is shown in green and contoured at 3.0σ. Electron density 2F_o–F_c is shown in blue contoured at 1.5σ and superimposed around sulfate and glycerol. Carbon atoms are shown in green (ligands), pink (pantoate pocket) and cyan (phosphate pocket), nitrogen in blue, oxygen in red, and sulfur in yellow. The figure was generated and rendered by using Pymol v. 0.99.^[35]

Figure 2

Analysis of dynamic combinatorial libraries. HPLC traces of DCLs with thiols 6 and 8a–h in a glutathione redox buffer after 24 h; A) with no protein, the disulfide conjugate between 6 and glutathione forming the major constituent, and B) with 200 μm M. tuberculosis pantothenate synthetase, showing amplification of thiol 6 and disulfide 14a.

Figure 3

Crystal structure of disulfide 14b bound in the active site of M. tuberculosis pantothenate synthetase. Omit electron density F_o–F_c is shown in green and contoured at 3.0σ around 14b. The ligand is shown as sticks with green carbons, and hydrogen-bonded water is shown as a red sphere. Key protein residues are shown with pink carbon atoms, nitrogen in blue, oxygen in red, and sulfur in yellow. The figure was generated and rendered by using Pymol v. 0.99.^[35]

Scheme 1

The pantoyladenylate intermediate 5 inspired the design of disulfides 7, which are formed by the dynamic exchange of a library comprised of thiols 8a–h and 6.

Scheme 2

Synthetic scheme for the synthesis of asymmetric disulfides and thioethers from thiol 6. a) diethyl azodicarboxylate, AcSH, PPh₃, THF; b) TFA, H₂O, 4 °C; c) NH₃, MeOH; d) i: NaOCl, ii: pyridine-2-thiol; e) CHCl₃, CH₃CO₂H; f) NaOMe, RSH; g) PPh₃, CBr₄; h) NaOMe, MeOH; i) TFA, H₂O, 4 °C.