A Fe2+-dependent self-inhibited state influences the druggability of human collagen lysyl hydroxylase (LH/PLOD) enzymes

Luigi Scietti; Elisabetta Moroni; Daiana Mattoteia; Marco Fumagalli; Matteo De Marco; Lisa Negro; Antonella Chiapparino; Stefano A Serapian; Francesca De Giorgi; Silvia Faravelli; Giorgio Colombo; Federico Forneris

doi:10.3389/fmolb.2022.876352

A Fe²⁺-dependent self-inhibited state influences the druggability of human collagen lysyl hydroxylase (LH/PLOD) enzymes

Front Mol Biosci. 2022 Aug 25:9:876352. doi: 10.3389/fmolb.2022.876352. eCollection 2022.

Affiliations

¹ The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.
² Consiglio Nazionale delle Ricerche, Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC-CNR), Milano, Italy.
³ Department of Chemistry, University of Pavia, Pavia, Italy.

Abstract

Multifunctional human collagen lysyl hydroxylase (LH/PLOD) enzymes catalyze post-translational hydroxylation and subsequent glycosylation of collagens, enabling their maturation and supramolecular organization in the extracellular matrix (ECM). Recently, the overexpression of LH/PLODs in the tumor microenvironment results in abnormal accumulation of these collagen post-translational modifications, which has been correlated with increased metastatic progression of a wide variety of solid tumors. These observations make LH/PLODs excellent candidates for prospective treatment of aggressive cancers. The recent years have witnessed significant research efforts to facilitate drug discovery on LH/PLODs, including molecular structure characterizations and development of reliable high-throughput enzymatic assays. Using a combination of biochemistry and in silico studies, we characterized the dual role of Fe²⁺ as simultaneous cofactor and inhibitor of lysyl hydroxylase activity and studied the effect of a promiscuous Fe²⁺ chelating agent, 2,2'-bipyridil, broadly considered a lysyl hydroxylase inhibitor. We found that at low concentrations, 2,2'-bipyridil unexpectedly enhances the LH enzymatic activity by reducing the inhibitory effect of excess Fe²⁺. Together, our results show a fine balance between Fe²⁺-dependent enzymatic activity and Fe²⁺-induced self-inhibited states, highlighting exquisite differences between LH/PLODs and related Fe²⁺, 2-oxoglutarate dioxygenases and suggesting that conventional structure-based approaches may not be suited for successful inhibitor development. These insights address outstanding questions regarding druggability of LH/PLOD lysyl hydroxylase catalytic site and provide a solid ground for upcoming drug discovery and screening campaigns.

Keywords: 2-2'-bipyridyl; Fe2+/2-oxoglutarate-dependent dioxygenases; cancer metastasis; collagen; lysyl hydroxylase (LH); molecular dynamics simulations; structure-based drug design.