Synthesis and Evaluation of a Non-Peptide Small-Molecule Drug Conjugate Targeting Integrin αVβ3

Abstract

An integrin αVβ3-targeting linear RGD mimetic containing a small-molecule drug conjugate (SMDC) was synthesized by combining the antimitotic agent monomethyl auristatin E (MMAE), an enzymatically cleavable Val-Ala-PABC linker with a linear conjugable RGD mimetic. The structure proposal for the conjugable RGD mimetic was suggested upon the DAD mapping analysis of a previously synthesized small-molecule RGD mimetic array based on a tyrosine scaffold. Therefore, a diversifying strategy was developed as well as a novel method for the partial hydrogenation of pyrimidines in the presence of the hydrogenolytically cleavable Cbz group. The small-molecule RGD mimetics were evaluated in an ELISA-like assay, and the structural relationships were analyzed by DAD mapping revealing activity differences induced by structural changes as visualized in dependence on special structural motifs. This provided a lead structure for generation of an SMDC containing the antimitotic drug MMAE. The resulting SMDC containing a linear RGD mimetic was tested in a cell adhesion and an in vitro cell viability assay in comparison to reference SMDCs containing cRGDfK or cRADfK as the homing device. The linear RGD SMDC and the cRGDfK SMDC inhibited adhesion of αVβ3-positive WM115 cells to vitronectin with IC50 values in the low µM range, while no effect was observed for the αVβ3-negative M21-L cell line. The cRADfK SMDC used as a negative control was about 30-fold less active in the cell adhesion assay than the cRGDfK SMDC. Conversely, both the linear RGD SMDC and the cRGDfK SMDC are about 55-fold less cytotoxic than MMAE against the αVβ3-positive WM115 cell line with IC50 values in the nM range, while the cRADfK SMDC is 150-fold less cytotoxic than MMAE. Hence, integrin binding also influences the antiproliferative activity giving a targeting index of 2.8.

RGD Peptidomimetic MMAE‐Conjugate Addressing Integrin αVβ3‐Expressing Cells with High Targeting Index**

“…In accordance with previous experience [30] the pentyl connector (n = 3) together with the DHI group show the best Chemistry-A European Journal…”

“…In comparison to the previously described benzoyl‐type RGD mimetics, [30] the general affinity for integrin α V β 3 was improved by introducing the sulfonamides, and even the selectivity over integrin α 5 β 1 could be improved (Table 3). In particular, the shorter RGD mimetics with a length of n=1–2 in combination with a benzoyl or para/meta‐hydroxy benzoyl substituent showed a significant affinity and selectivity for integrin α 5 β 1 as the connector length decreased [30] . This effect is not observable for the sulfonamides, which avoid repulsion between the aromatic residue and the binding pocket due to the tetrahedral sulfonamide.…”

“…[29] Self-immolative peptide linker for payload attachment In agreement with previously described SMDCs, a linker that can be enzymatically cleaved by proteases has been synthesized. [30] After integrin-mediated endocytosis, the conjugate is supposed to end up in the lysosome, where proteases such as cathepsins are present. Cathepsins are suitable targets for payload release because they are overexpressed in some cancer cells.…”

Antibody-Drug Conjugates Containing Payloads from Marine Origin

“…Although it is out of the scope of the present review, it is important to mention alternative tactics that are being explored with the auristatins with the objective of targeting cancer cells in a highly selective manner by means of platforms different from antibodies recognized by tumor antigens. Among these alternative devices, it is worth highlighting low-molecular-weight ligands for antigens such as the prostate-specific membrane antigen (PSMA) [ 129 ] or integrin ανβ 3 [ 130 ], aptamers [ 131 ], gold nanoclusters [ 132 ] or radiolabeled proteins [ 133 ].…”

Evaluation of a Radio-IMmunoStimulant (RIMS) in a Syngeneic Model of Murine Prostate Cancer and ImmunoPET Analysis of T-cell Distribution

“…To this end, we introduce the Radio-IMmunostimulant (RIMS), which is a single chemical entity comprised of 4 components (Figure ): (1) a PSMA targeting motif with an optimized naphthyl linker for enhanced binding affinity; (2) the metal chelator DOTA that coordinates the β – particle emitting radiometal 177 Lu, which causes DNA scission and cell death. Importantly, due to the range of the β – particle (mean range: 0.7 mm), 177 Lu can kill nontarget cancer cells (PSMA low or PSMA(−)) via the bystander effect, which by itself does not provide significant heterogeneous tumor regression. − Additionally, the RIMS construct also delivers and releases a (3) small molecule immunostimulant payload mimicking viral ssRNA that activates toll-like receptors 7 and 8 (TLR 7/8) − following cathepsin mediated , scission of (4) an enzymatically cleavable peptide sequence − attached to a self-immolative linker . In this work, we demonstrate the chemical assembly, characterization, in vitro and in vivo validation of the RIMS paradigm in a syngeneic animal model of prostate cancer, supported by monitoring immune cell recruitment with 89 Zr-immuno-PET and a direct comparison with the recently FDA approved therapy for mCRPC, 177 Lu-PSMA-617.…”