Rational design of NT-PSMA heterobivalent probes for prostate cancer theranostics

Abstract

Targeting the prostate-specific membrane antigen (PSMA) with radiopharmaceuticals for imaging and/or therapy has demonstrated significant advancement in the management of prostate cancer patients. However, PSMA targeting remains unsuccessful in prostate cancers with low expression of PSMA, which account for 15% of cases. The neurotensin receptor-1 (NTS₁) has been highlighted as a suitable oncotarget for imaging and therapy of PSMA-negative prostate cancer lesions. Therefore, heterobivalent probes targeting both PSMA and NTS₁ could improve the prostate cancer management. Herein, we report the development of a branched hybrid probe (JMV 7489) designed to target PSMA and/or NTS₁ bearing relevant pharmacophores and DOTA as the chelating agent. The new ligand was synthesized with a hybrid approach, which includes both syntheses in batch and in the solid phase. Saturation binding experiments were next performed on HT-29 and PC3-PIP cells to derive K _d and B _max values. On the PC3-PIP cells, [⁶⁸Ga]Ga-JMV 7489 displayed good affinity towards PSMA (K _d = 53 ± 17 nM; B _max = 1393 ± 29 fmol/10⁶ cells) in the same range as the corresponding reference monomer. A lower affinity value towards NTS₁ was depicted (K _d = 157 ± 71 nM; B _max = 241 ± 42 fmol/10⁶ cells on PC3-PIP cells; K _d = 246 ± 1 nM; B _max = 151 ± 44 fmol/10⁶ cells on HT-29 cells) and, surprisingly, it was also the case for the corresponding monomer [⁶⁸Ga]Ga-JMV 7089. These results indicate that the DOTA macrocycle and the linker are critical elements to design heterobivalent probes targeting PSMA and NTS₁ with high affinity towards NTS₁.

Chart 1. Structures of [¹⁷⁷Lu]Lu-PSMA-617, [⁶⁸Ga]Ga-PSMA-11, SR-48692, SR142948A, and probes targeting NTS₁. The structure of DOTA was solely reported in [¹⁷⁷Lu]Lu-PSMA-617.

Chart 2. Structure and biological activity of compound 1. NT and its modified analogues with biological activity towards NTS₁. Structure of the branched heterobivalent probes JMV 7489 and JMV 7089: DOTA, β-Ala linkers, Glu, O-(carboxymethyl)-Tyr-Phe fragments, and PSMA binding regions are reported in orange, gray, red, green, and light blue, respectively. The NT arm is reported as the amino acid sequence.

Scheme 1. Reagents and conditions: a) Fmoc-β-Ala-OH, HATU, DIPEA, DMF, 0 °C, then rt, on; b) methyl bromoacetate, K₂CO₃, ACN, rt, on; c) NaOH, CaCl₂, iPr-OH/H₂O 7:3, rt, TLC monitoring, d) HATU, DMF, 0 °C, then rt, on; e) 20% piperidine in DMF, 1 h, rt; f) 20% piperidine in DMF, 5 min × 3, rt, in an SPPS syringe; g) HATU, DIPEA, appropriate Fmoc-AA-OH, 1 h, rt, in an SPPS syringe; h) DCM, TFE, AcOH (8 : 1 : 1), 4 h, rt, in an SPPS syringe. All reactions were performed in a round bottom flask unless otherwise specified.

Scheme 2. Reagents and conditions: a) in a round bottom flask: triphosgene, TEA, DCM, 30 min, 0 °C, then rt; b) 20% piperidine in DMF, 5 min × 3, then 13 in a round bottom flask; c) 20% piperidine in DMF, 5 min × 3, rt; d) appropriate Fmoc-AA-OH, HATU, DIPEA, DMF, 1 h, rt; e) TFA/TIS/H₂O (95/2.5/2.5), 7 h, rt. All reactions were performed in an SPPS syringe unless otherwise specified.

Fig. 1. Saturation binding curves of [⁶⁸Ga]Ga-JMV 7489 showing specific binding at PSMA performed on PC3-PIP cells (squares) and specific binding at NTS₁ on HT-29 cells (circles). The NTS₁-specific binding performed on PC3-PIP is not shown for easier reading.