Structure-Guided Drug Design of 6-Substituted Adenosine Analogues as Potent Inhibitors of Mycobacterium tuberculosis Adenosine Kinase

Abstract

Mycobacterium tuberculosis adenosine kinase (MtbAdoK) is an essential enzyme of Mtb and forms part of the purine salvage pathway within mycobacteria. Evidence suggests that the purine salvage pathway might play a crucial role in Mtb survival and persistence during its latent phase of infection. In these studies, we adopted a structural approach to the discovery, structure-guided design, and synthesis of a series of adenosine analogues that displayed inhibition constants ranging from 5 to 120 nM against the enzyme. Two of these compounds exhibited low micromolar activity against Mtb with half maximal effective inhibitory concentrations of 1.7 and 4.0 μM. Our selectivity and preliminary pharmacokinetic studies showed that the compounds possess a higher degree of specificity against MtbAdoK when compared with the human counterpart and are well tolerated in rodents, respectively. Finally, crystallographic studies showed the molecular basis of inhibition, potency, and selectivity and revealed the presence of a potentially therapeutically relevant cavity unique to the MtbAdoK homodimer.

Figure 1

Positions of interest for structure-guided drug design and tool compounds utilized in crystallography studies. (a) Adenosine bound to the active site of MtbAdoK (PDB ID 2PKM). (b) Tool compounds used to explore the chemical space surrounding the positions highlighted in (a).

Figure 2

Crystal structure complexes of compounds 2–5. (a) 2 bound to the active site of MtbAdoK. (b) 3 bound to the active site of MtbAdoK. (c,d) N7-substituents of compounds 2–3 are buried in an active site pocket composed of residues from chain A and chain B. (e) Chimney-like cavity observed above position N6 formed by residues from chain A and chain B. (f) 4 bound to the active site of MtbAdoK. (g) Methylmercapto group of 4 is accommodated in a compound-induced pocket formed by the movement of Arg176 (red). (h) Chimney-like cavity observed in the cocrystal structures of 2 and 3 is blocked by the conformational change of Arg176 (red). (i) 5 bound to the active site of MtbAdoK. In all cases, chain A residues are colored by heteroatom and chain B residues are color magenta.

Figure 3

Crystal structure of the MtbAdoK-6 complex. (a) Chemical structure of compound 6. (b) 6 bound to the active site of MtbAdoK. (c) Superimposition of the cocrystal structures of compound 6 (gray) and adenosine (blue). (d) Surface representation of residues forming the pocket where the thiophene group gets accommodated. Chain B is colored magenta, Arg176 is colored red, and chain A is colored by heteroatom. (e) Arg176 adopts a different conformation in the cocrystal structure of 4 (blue) when compared with the cocrystal structure of 6 (gray).

Scheme 1. Synthesis of Adenosine Analogues

Reagents and conditions: (a) TBSCl, imidazole, DCM, 15 °C, 17 h, 99%; (b) 1-phenylpiperazine or 1-([1,1′-biphenyl]-4-yl)piperazine or 1-(4-bromophenyl)piperazine, DIEA, EtOH, 70–80 °C, 17 h, 13–90%; (c) ArB(OH)₂, K₃PO₄, XPhos Pd G2, THF/H₂O, 70 °C, 17 h, 49–87%; (d) TFA, THF/H₂O, 15–25 °C, 2–17 h, 13–67%; (e) 4,4,5,5-tetramethyl-2-(4-(phenylethynyl)phenyl)-1,3,2-dioxaborolane, K₃PO₄, XPhos Pd G2, THF, 70 °C, 12 h, 95% or 4-ethynyl-1,1′-biphenyl, Cs₂CO₃, CuI, XPhos Pd G2, CH₃CN, 90 °C, 17 h, 27%; (f) 1-([1,1′-biphenyl]-4-yl)piperazine, Cs₂CO₃, 1-([1,1′-biphenyl]-4-yl)piperazine, RuPhos Pd G2, tert-amyl-OH, 100 °C, 17 h, 50%; and (g) NH₄OH, MeOH, 15 °C, 24 h, 21%.

Figure 4

Crystal structure of the MtbAdoK-7 complex. (a) 7 bound to the active site to MtbAdoK. Chain A is colored gray and chain B is colored magenta. (b) The bulky substituent of 7 (gray) forces the compound to bind in a different orientation with respect to adenosine (blue; PDB ID 2PKM). (c) The bulky substituent forces Arg176 to open the cavity while the distal benzene ring is reoriented back into the active site groove that is formed by residues of chain A (heteroatom) and chain B (magenta).

Figure 5

Summary of SAR results for compounds 8–17. Compound 7 bound to the active site of MtbAdoK. Based on the MtbAdoK-7 complex, compounds 8–17 where synthesized. Chain A is colored white, and chain B is colored magenta.

Figure 6

Crystal structure of the MtbAdoK-17 complex. (a) 17 bound to the active site of MtbAdoK. (b) The large bulky substitution gets accommodated in the “chimney-like” cavity. (c) Compound 17 (gray) binds in a different orientation with respect to adenosine (blue). (d) Superimposition of crystal structure complexes of 17 (gray) and 7 (blue). Chain B residues forming the distal part of the “chimney-like” cavity colored magenta while chain A residues are colored by heteroatom.

Figure 7

Preliminary pharmacokinetics of compound 18. (a) Oral PK study conducted at 10 mg·kg^–1 utilizing 9 Swiss Webster Female mice with three animal bleeds per time point. Maximum plasma concentration of 0.13 ± 0.02 μg·mL^–1 observed at 4 h following gavage. The AUC_0–24h of 1.8 μg·h·mL^–1 was calculated using the linear trapezoid rule method. (b) Snapshot PK studies conducted from plasma samples taken from tolerability mice at 50, 100, and 200 mg·kg^–1 following first initial dose. 10 mg·kg^–1 data included for comparison. (c) C_max, t_max, and AUC of compound 18 at four dose levels.