METTL3-METTL14 Inhibition: A Novel Therapeutic Target for Acute Lung Injury

Abstract

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are acute, inflammatory clinical syndromes characterized by poor oxygenation and diffuse pulmonary infiltrates. This syndrome is associated with microvascular endothelial dysfunction and subsequent pulmonary hypertension and may ultimately lead to mortality without rigorous and acute clinical intervention. Over the years, many attempts have been made to detect novel therapeutic avenues for research without much success. The urgency for the discovery of novel therapeutic agents has become more pronounced recently given the current pandemic infection of coronavirus disease 2019 (COVID-2019), still ongoing at the time that this dissertation is being written. N6-methyladenosine (m6A) is the most common methylation modification in mammalian messenger RNA (mRNA) and noncoding RNAs that plays a role in the regulation of gene expression. Deregulation of m6A methylation has been implicated in many human diseases and exploitation of the methylation process may possess utility against ALI. In chapter I, we review the current landscape of literature regarding ALI and ARDS etiology, pathophysiology, and therapeutics and present a potential role of m6A methylation. Additionally, we will establish the axiomatic principles of m6A methylation to provide a framework for chapters II-IV. In chapter II, we explore gene expression in response to ALI-related stimuli. We confirmed METTL3 as a vital component of m6A up-regulation following LPS treatment and demonstrated alleviation of endothelial barrier dysfunction by METTL3 knockdown-suggesting that METTL3 inhibition may possess therapeutic utility for ALI. We used a known METTL3 inhibitor (STM) to selectively inhibit METTL3’s demethylation which induced the increase of m6A mRNA methylation level in human pulmonary artery endothelial cells (HPAECs). METTL3 inhibition reduced m6A methylation levels following LPS treatment and reduced ICAM1 levels. Additionally, LPS-induced responses in endothelial barrier regulation were attenuated by METTL3 knockdown. In chapter III, we continue exploring gene expression deregulation in response to ALI-related stimuli. We present our findings from a similar m6A methylation project focusing on the effects of particulate matter and airway epithelium. Our group has experience studying the effects of PM2.5 on cardiopulmonary systems and decided to pursue a side project using our m6A methylation platform to examine whether PM2.5 can produce this functional and robust post-transcriptional regulation. We confirmed an increase in m6A methylation activity following PM2.5 treatment which correlated with increased METTL3 and METTL14 protein expression. METTL3 knockdown greatly attenuated PM2.5 -induced m6A methylation and decreased protein expression of ICAM1. Moreover, METTL3 knockdown decreased the protein expression of ERK and up-regulated protein expression of p-JNK and JNK, highlighting a potential link between m6A methylation and the MAPK signaling pathway. Lastly, we observed that PM2.5 increased mRNA expression of both MAP3k8 and CXCL5 in A549 cells and that TNF-α secretion increased in PM2.5-treated cells. In chapter IV, we sought to identify a small molecule inhibitor of METTL3 using a novel virtual drug discovery campaign. Our strategy was to identify ligands by virtually screening molecular libraries for modulators of the RNA methyltransferase METTL3-14-WTAP complex and then to characterize their binding properties as well as effects on enzymatic activity with the intention of ultimately choosing a lead compound for animal studies of ALI. Using the crystal structure of the METTL3-METTL14 complex, we searched databases compromising of almost 8 million compounds to identify a small molecule compound with favorable docking to the SAM binding pocket. Ultimately, 24 compounds passed visual inspection following our cluster-based selection. Twenty-three small molecule compounds with favorable glide scores were procured and chosen to undergo METTL3-14 binding affinity and kinetics studies using surface plasmon resonance (SPR). Of the 23 compounds, 6 demonstrated favorable binding to the METTL3-14 complex and underwent a bioluminescence methyltransferase activity assay to determine whether any compounds contained METTL3-14 inhibitory activity. Two compounds, ZRF001 and Z70895572, exhibited concentration-dependent methyltransferase inhibitory activity with ZRF001 demonstrating IC50 value in the low uM range. Compound ZRF001 was ultimately chosen as our lead compound for in vitro studies of ALI. In conclusion, METTL3 is a hub of pro-inflammatory gene expression regulation in ALI, and using a modern drug discovery strategy will identify new and effective ALI drug candidates targeting METTTL3.