Hybrid topoisomerase I and HDAC inhibitors as dual action anticancer agents

Abstract

Recent studies have shown that HDAC inhibitors act synergistically with camptothecin derivatives in combination therapies. To exploit this synergy, new hybrid molecules targeting simultaneously topoisomerase I and HDAC were designed. In particular, a selected multivalent agent containing a camptothecin and a SAHA-like template showed a broad spectrum of antiproliferative activity, with IC50 values in the nanomolar range. Preliminary in vivo results indicated a strong antitumor activity on human mesothelioma primary cell line MM473 orthotopically xenografted in CD-1 nude mice and very high tolerability.

Fig 1. Structures of representative HDAC and topoisomerase I inhibitors.

Fig 2. Synthesis of compounds 3a-d.

Reagents and conditions: a) for 2a-c: HCl·H₂NO(CH₂)_nCOOH, acetic acid, 80 °C, 3h; for 2d: HCl·H₂NO(CH₂)_nCOOH, EtOH, pyridine, reflux, 4h; 2a: 90%, 2b: 73%, 2c: 70%, 2d:75%; b) for 3a: DCC, NHS, HCl·NH₂OH, rt, 48h, 10%; for 3b-d, WSC, HOBt, DMF, NH₂OH·HCl, TEA, rt, 1-8h, 3b: 61%, 3c: 45%, 3d: 73%.

Fig 3. Synthesis of compound 10.

Reagents and conditions: a) N-hydroxyphthalimide, PPh₃, DIAD, THF, 63%; b) TFA, CH₂Cl₂, 94%; c) TrSCH₂COOH, EDCI, DMAP, TEA, CH₂Cl₂, 53%; d) NH₂NH₂, CH₃OH, 83%; e) 7-formylcamptothecin, EtOH, 91%; f) TFA, Et₃SiH, CH₂Cl₂, 80%.

Fig 4

(Left)—Schematic representation of the proposed top-score binding mode for compound 3d in the DNA-Topoisomerase-I complex. Topoisomerase-I is represented as Backbone Ribbon colored from blue (N-term) to red (C-term), while nucleic acids are represented as an arrow along the backbone pointing toward the C3'-end, and sugar groups and bases as boxes (A in red, C in violet, G in green and T in blue). The 3d ligand and the catalytic tyrosine (CT) are rendered in CPK (3d in red and CT in blue). (Right)—Hypothetical binding mode for the Top-score conformation of 3d in complex with DNA-Topoisomerase-I, with the aminoacids side chains relevant to the discussion rendered in line and the ligands in stick. Hydrogen bonds are shown as green dotted lines.

Fig 5

(Left)—Solvent Accessible Surface (SAS) representation of HDAC bond to 3d (in stick). The portions of molecule interacting with the binding site is delimited by the oxime group, located near the entrance of the cavity. (Right)—Binding mode of the top-score conformation of 3d to HDAC-II, with the aminoacids side chains relevant to the discussion rendered in line and the ligand in stick. Hydrogen bonds are shown as green dotted lines.

Fig 6

(A)–Superimposition of the 3d (in red) and CPT (blue) structures in the catalytic site of Top1. (B)—Overlap of 3d (red) and SAHA (blue) within the HDAC-II catalytic site.

Fig 7. Effects of compounds 3b, 3d, SN38 and SAHA (reference compounds) on acetylation of tubulin and histone H4 in NCI-H460 cells.

Cells were treated for 24 h with SN38 (0.05 μM), SAHA (5 μM), 3b and 3d (0.125–0.2–0.5 μM). Cells were lysed in 0.5% NP-40 and 40 μg of total proteins were loaded on NuPAGE 4–12% (Invitrogen), transferred to a nitrocellulose paper and probed with specific antibodies. Acetylated Histone H4 (Ac-H4); Histone H4; Acetylated Tubulin (Ac-H4); Tubulin.

Fig 8. Effects of compound 3d, SN38 and SAHA (reference compounds) on Top1, H2A.X, p21 (A), CycD1, p16, p53 (B) in NCI-H460 cells.

H460 cells were treated for 24 h with SAHA (5 μM), SN38 (0.05 μM), and 3d (0.125–0.2–0.5μM). Cells were lysed in 0.5% NP-40 and 40 μg of total proteins were loaded on NuPAGE 4–12% (Invitrogen), transferred to a nitrocellulose paper and probed with specific antibodies.

Fig 9. Efficacy of 3d in an orthotopic xenograft model of human epithelioid mesothelioma.

Bioluminescence signal from mice injected with MM473 expressing luciferase at day 15 (first treatments) and day 40 (8 days after the last treatment).

Fig 10. Efficacy of 3d in an orthotopic xenograft model of human epithelioid mesothelioma.

a) bioluminescence signal of experimental groups throughout the experimental period; b) body weight curve. Statistical analysis performed using non parametric Mann-Whitney test (U) 3d groups versus Vehicle *p<0,05.