Preclinical Development of PNT6555, a Boronic Acid-Based, Fibroblast Activation Protein-α (FAP)-Targeted Radiotheranostic for Imaging and Treatment of FAP-Positive Tumors.

The overexpression of fibroblast activation protein-α (FAP) in solid cancers relative to levels in normal tissues has led to its recognition as a target for delivering agents directly to tumors. Radiolabeled quinoline-based FAP ligands have established clinical feasibility for tumor imaging, but their therapeutic potential is limited due to suboptimal tumor retention, which has prompted the search for alternative pharmacophores. One such pharmacophore is the boronic acid derivative N-(pyridine-4-carbonyl)-d-Ala-boroPro, a potent and selective FAP inhibitor (FAPI). In this study, the diagnostic and therapeutic (theranostic) potential of N-(pyridine-4-carbonyl)-d-Ala-boroPro-based metal-chelating DOTA-FAPIs was evaluated. Methods: Three DOTA-FAPIs, PNT6555, PNT6952, and PNT6522, were synthesized and characterized with respect to potency and selectivity toward soluble and cell membrane FAP; cellular uptake of the Lu-chelated analogs; biodistribution and pharmacokinetics in mice xenografted with human embryonic kidney cell-derived tumors expressing mouse FAP; the diagnostic potential of 68Ga-chelated DOTA-FAPIs by direct organ assay and small-animal PET; the antitumor activity of 177Lu-, 225Ac-, or 161Tb-chelated analogs using human embryonic kidney cell-derived tumors expressing mouse FAP; and the tumor-selective delivery of 177Lu-chelated DOTA-FAPIs via direct organ assay and SPECT. Results: DOTA-FAPIs and their natGa and natLu chelates exhibited potent inhibition of human and mouse sources of FAP and greatly reduced activity toward closely related prolyl endopeptidase and dipeptidyl peptidase 4. 68Ga-PNT6555 and 68Ga-PNT6952 showed rapid renal clearance and continuous accumulation in tumors, resulting in tumor-selective exposure at 60 min after administration. 177Lu-PNT6555 was distinguished from 177Lu-PNT6952 and 177Lu-PNT6522 by significantly higher tumor accumulation over 168 h. In therapeutic studies, all 3 177Lu-DOTA-FAPIs exhibited significant antitumor activity at well-tolerated doses, with 177Lu-PNT6555 producing the greatest tumor growth delay and animal survival. 225Ac-PNT6555 and 161Tb-PNT6555 were similarly efficacious, producing 80% and 100% survival at optimal doses, respectively. Conclusion: PNT6555 has potential for clinical translation as a theranostic agent in FAP-positive cancer.

Detecting Functional and Accessible Folate Receptor Expression in Cancer and Polycystic Kidneys.

Folate-based small molecule drug conjugates (SMDCs) are currently under development and have shown promising preclinical and clinical results against various cancers and polycystic kidney disease. Two requisites for response to a folate-based SMDC are (i) folate receptor alpha (FRα) protein is expressed in the diseased tissues, and (ii) FRα in those tissues is accessible and functionally competent to bind systemically administered SMDCs. Here we report on the development of a small molecule reporter conjugate (SMRC), called EC2220, which is composed of a folate ligand for FRα binding, a multilysine containing linker that can cross-link to FRα in the presence of formaldehyde fixation, and a small hapten (fluorescein) used for immunohistochemical detection. Data show that EC2220 produces a far greater IHC signal in FRα-positive tissues over that produced with EC17, a folate-fluorescein SMRC that is released from the formaldehyde-denatured FRα protein. Furthermore, the extent of the EC2220 IHC signal was proportional to the level of FRα expression. This EC2220-based assay was qualified both in vitro and in vivo using normal tissue, cancer tissue, and polycystic kidneys. Overall, EC2220 is a sensitive and effective reagent for evaluating functional and accessible receptor expression in vitro and in vivo.

Diastereo- and enantioselective three-component coupling approach to highly substituted pyrrolidines.

The enantioselective synthesis of substituted pyrrolidines through a mild Lewis-acid catalyzed three-component coupling reaction between picolinaldehyde, amino acids, and activated olefins is reported. The reaction uses low catalyst loadings of commercially available chiral diamines and copper triflate proposed to self-assemble in conjunction with the chelating aldehydes, 4-substituted-2-picolinaldehydes or 4-methylthiazole-2-carboxaldehyde, to generate a catalyst complex. A model is provided to explain how this complex directs enantioselectivity. This work represents a significant advance in the ease, scope, and cost of producing highly substituted, enantioenriched pyrrolidines.

Simplifying pyridoxal: practical methods for amino acid dynamic kinetic resolution.

Metal complexes of picolinaldehyde are identified as low-cost and environmentally benign catalysts, providing high reaction rates and turnovers for the racemization of amino acids. These pyridoxal surrogates demonstrate activity toward a variety of amino acid esters. Applications to chemoenzymatic dynamic kinetic resolutions provide access to amino acids in high yields and with excellent enantioselectivities, demonstrating their compatibility with protease-mediated transformations.