Structure-based design of selective and potent G quadruplex-mediated telomerase inhibitors

Abstract

The telomerase enzyme is a potential therapeutic target in many human cancers. A series of potent inhibitors has been designed by computer modeling, which exploit the unique structural features of quadruplex DNA. These 3,6,9-trisubstituted acridine inhibitors are predicted to interact selectively with the human DNA quadruplex structure, as a means of specifically inhibiting the action of human telomerase in extending the length of single-stranded telomeric DNA. The anilino substituent at the 9-position of the acridine chromophore is predicted to lie in a third groove of the quadruplex. Calculated relative binding energies predict enhanced selectivity compared with earlier 3,6-disubstituted compounds, as a result of this substituent. The ranking order of energies is in accord with equilibrium binding constants for quadruplex measured by surface plasmon resonance techniques, which also show reduced duplex binding compared with the disubstituted compounds. The 3,6,9-trisubstututed acridines have potent in vitro inhibitory activity against human telomerase, with EC(50) values of up to 60 nM.

Figure 1

Structures of compounds 1–4.

Figure 2

Synthetic schemes for the 3,6,9-trisubstituted acridine derivatives 3 and 4. The individual steps involved (i) KNO₃/H₂SO₄, (ii) CrO₃, AcOH, reflux, (iii) Zn/HCl, 90–100°C, (iv) 3-CPC, reflux, (v) NHR₂, EtOH, NaI, reflux, (vi) HCl, (vii) POCl₃, reflux, and (viii) H₂NPhNR₂, CHCl₃, rt.

Figure 3

(a) View of the simulated structure of compound 1 bound to the high-affinity binding site in the human quadruplex structure formed from the sequence d[AG₃(TTAG₃)₃], looking onto the terminal guanine quartet. (b) View of the complex with compound 3 bound in the same site, at the end of a 1-ns molecular dynamics simulation. (c) Overall view of the complex with compound 3 (shown as a solvent-accessible surface, with nitrogen atoms highlighted in blue). The two sodium atoms in the central channel of the G quartet are shown in mauve.

Figure 4

(a) A set of SPR sensorgrams for binding of compound 3 to the human G quadruplex at 25°C is shown. The unbound ligand concentrations in the flow solution were 0, 5, 10, 20, 40, 60, 80, 100, 200, and 600 nM from the lowest curve to the top curve. The lines are linear best fits to the steady-state RU (response units) values, which are directly proportional to the amount of bound compound, and were used to determine the RU for each free ligand concentration. (b) The RU values from the steady-state region from a plotted versus the unbound compound 3 (■) concentration. Experiments similar to those in a also were conducted for compound 1 (●) and curves for the two derivatives are shown. The lines were obtained by nonlinear least-square fits of the data. Results for compound 4 were similar to those for 3. Equilibrium constants for all compounds are given in Table 3.