Associate Professor, Chemistry
University of Kansas
Crystallization and inhibitor screening of lysyl oxidase-like 2 (2018-19)
Cardiac, liver and lung fibrosis are characterized by thickening and stiffening of tissues within these organs. Lysyl oxidase-like 2 (LOXL2) has shown to promote excessive accumulation of the extracellular matrix (ECM) via catalyzing irreversible crosslinking of collagen and activation of fibroblasts. The long-term objective of the proposed research is to develop specific inhibitors of LOXL2 as such inhibitors could be further developed into a targeted therapy for fibrosis.
This project aims to 1) screen crystallization conditions for minimally N-glycosylated recombinant LOXL2s (rLOXL2s, mature and precursor forms) at the KU COBRE-PSF Protein X-ray Structure Core and 2) identify fragments that bind to the active site of the precursor form of LOXL2 by screening of three fragment libraries using a surface plasmon resonance spectroscopy at the KU COBRE-PSF Fragment Screening Core and followed by activity-based assay to select fragments that inhibit biogenesis of the LTQ cofactor.
For rLOXL2 proposed in Aim 1, we established protocols to isolate a mature form of rLOXL2 where the first two of the four SRCR domains at the Nterminal are truncated (Δ1-2SRCR-LOXL2) with only one GlcNac residue attached to Asn455 and Asn644, through the support from COBRE-PSF 2017 Pilot Project, The mature rLOXL2 contains nearly stoichiometric amount of the LTQ cofactor and that is covalently stabilized by treatment with 2-hydrazinopyridine. We will apply the method to produce the precursor form (no LTQ, Zn2+-bound) of Δ1-
2SRCR-LOXL2 with only one GlcNAc.
For rLOXL2 proposed in Aim2, we will use the precursor form (no LTQ, no metal) of Δ1-2SRCR-LOXL2 with normal N-glycosylation. The successful completion of Aim 1 will yield crystals suitable for X-ray diffraction analysis, and upon successful completion of aim 2, we will identify fragments that bind to the LOXL2 active site to inhibit the LTQ biogenesis. Such preliminary results will greatly strengthen the competitive renewal of a R01 entitled, “Understanding the roles of PTMs in modulating molecular functions of LOXL2 in breast cancer cells” in early 2019.
Screening of crystallization conditions of human LOXL2 and fragment library screening of its specific inhibitors (2017-18)
Lysyl oxidase-like 2 (LOXL2) has been considered as a therapeutic target for lung and liver fibrosis and aggressive/invasive tumors (breast, colon, esophageal, gastric, liver, lung and renal). However, its protein crystal structure has not been resolved due to the difficulties in producing recombinant LOXL2 suitable to conduct such study. In this study, we will take advantage of our recent success in isolating highly pure and active recombinant LOXL2 to conduct screening of its initial crystallization conditions and fragment-based ligand identification of LOXL2.
LOXL2 is highly upregulated in these diseases, and suppression of LOXL2 by shRNA or a specific antibody can slow fibrosis and tumor progression in mice. The long-term objective of the proposed research is to identify specific and selective inhibitors of LOXL2. Such inhibitors can be used for functional probes of LOXL2 in progression of diseases above mentioned and lead compounds for future drug development. Currently, there is no structural information available for LOXL2 or other four members of the lysyl oxidase (LOX)- family of proteins. This is because all of the currently available recombinant LOXL2s (rLOXL2s) and LOX(L)s are not pure/homogeneous. Consequently, very limited fundamental biochemical and biological characterization of LOXL2 has been conducted, and specific inhibitors for LOXL2 has not been identified.
The PI’s lab has recently succeeded in establishing a protocol for isolating highly pure (> 95%) and active rLOXL2 containing fully-biogenized lysine tyrosylquinone (LTQ) cofactor. With this highly quality of rLOXL2 in hands, this project aims to 1) screen crystallization conditions for rLOXL2 at KU COBRE-PSF Protein Xray Structure Core and 2) identify ligands by screening of three fragment libraries (total of 832 compounds) by surface plasmon resonance spectroscopy at KU COBRE-PSF Fragment Screening Core. The successful completion of Aim 1 will yield a crystal suitable for X-ray diffraction analysis, and such preliminary results will greatly strengthen the competitive renewal of the current R01. Upon the successful completion of Aim 2, the first series of LOXL2 ligands will be identified. The results will be compared with the 25 small molecules identified through HTS analysis, and taken together those results will provide building blocks for the future structure-based drug design.