Beyond the active site: Discovery of novel PYCR1 allosteric binding sites via structure-oriented fragment crystallographic screening

Pyrroline-5-carboxylate reductase 1 (PYCR1), a member of the PYCR enzyme family, plays a pivotal role in the proline biosynthesis pathway. Enzymes involved in proline metabolism have been increasingly recognized as key regulators of cancer cell metabolic reprogramming, contributing to enhanced proliferation, survival, metastasis, and resistance to stress. Overexpression of PYCR1 has been consistently reported across multiple malignancies, including hepatocellular carcinoma, breast cancer, gastric cancer, bladder cancer, and lung cancer, as well as in fibrotic disorders. These observations highlight PYCR1 as an attractive therapeutic target for oncology drug discovery. Despite its therapeutic potential, PYCR1 remains an understudied target, with limited data available on active small-molecule ligands and binding site characterization. Consequently, the discovery of novel chemical probes is critical both for validating PYCR1's role in disease pathology and for initiating structure-based drug design campaigns. To this end, a fragment-based screening campaign was initiated using a focused subset of carboxylic acid-containing fragments from the Enamine fragment library. The subset was rationally designed through molecular docking of fragment-like, carboxylic acid-containing compounds from Enamine's in-stock collection, targeting the active site of PYCR1. A total of 37 fragments were selected and screened via X-ray crystallography. This screen yielded eight fragments with confirmed binding to the PYCR1 active site, as determined by co-crystal structures. Among these, three compounds demonstrated inhibitory activity in biochemical assays, with IC₅₀ values ranging from 29 μM to 5.4 mM. Encouraged by these results, a follow-up screen was conducted using 22 fragment-like compounds containing bioisosteres of the carboxylic acid functional group (e.g., tetrazoles, oxadiazoles) and sulfonic acid groups, to explore alternative chemical scaffolds. This second crystallographic screen identified one additional active-site binder. Importantly, at least two previously uncharacterized allosteric binding sites were discovered, both of which showed a measurable inhibitory effect on PYCR1 enzymatic activity. These findings open promising new avenues for the development of non-classical inhibitors targeting PYCR1.