A peptide array pipeline for the development of Spike-ACE2 interaction inhibitors.

In humans, coronaviruses are the cause of endemic illness and have been the causative agents of more severe epidemics. Most recently, SARS-CoV-2 was the causative agent of the COVID19 pandemic. Thus, there is a high interest in developing therapeutic agents targeting various stages of the coronavirus viral life cycle to disrupt viral propagation. Besides the development of small-molecule therapeutics that target viral proteases, there is also interest molecular tools to inhibit the initial event of viral attachment of the SARS-CoV-2 Spike protein to host ACE2 surface receptor. Here, we leveraged known structural information and peptide arrays to develop an in vitro peptide inhibitor of the Spike-ACE2 interaction. First, from previous co-crystal structures of the Spike-ACE2 complex, we identified an initial 24-residue long region (sequence: STIEEQAKTFLDKFNHEAEDLFYQ) on the ACE2 sequence that encompasses most of the known contact residues. Next, we scanned this 24-mer window along the ACE2 N-terminal helix and found that maximal binding to the SARS-CoV-2 receptor binding domain (CoV2-RBD) was increased when this window was shifted nine residues in the N-terminal direction. Further, by systematic permutation of this shifted ACE2-derived peptide we identified mutations to the wildtype sequence that confer increased binding of the CoV2-RBD. Among these peptides, we identified binding peptide 19 (referred to as BP19; sequence: SLVAVTAAQSTIEEQAKTFLDKFI) as an in vitro inhibitor of the Spike-ACE2 interaction with an IC50 of 2.08 ± 0.38 µM. Overall, BP19 adds to the arsenal of Spike-ACE2 inhibitors, and this study highlights the utility of systematic peptide arrays as a platform for the development of coronavirus protein inhibitors.

SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation-Phosphorylation Cross-talk.

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy. SIGNIFICANCE: SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy. This article is highlighted in the In This Issue feature, p. 2007.

Investigating radical pair reaction dynamics of B12 coenzymes 1: Transient absorption spectroscopy and magnetic field effects.

B12 coenzymes are vital to healthy biological function across nature. They undergo radical chemistry in a variety of contexts, where spin-correlated radical pairs can be generated both thermally and photochemically. Owing to the unusual magnetic properties of B12 radical pairs, however, most of the reaction and spin dynamics occur on a timescale (picoseconds-nanoseconds) that cannot be resolved by most measurement techniques. Here, we describe a method that combines femtosecond transient absorption spectroscopy with magnetic field exposure, which enables the direct scrutiny of such rapid processes. This approach should provide a means by which to investigate the apparently profound effect protein environments have on the generation and reactivity of B12 radical pairs.

Aberrant RNA methylation triggers recruitment of an alkylation repair complex.

Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.

Lysine Methyltransferases Signaling: Histones are Just the Tip of the Iceberg.

Protein lysine methylation is a functionally diverse post-translational modification involved in various major cellular processes. Lysine methylation can modulate proteins activity, stability, localization, and/or interaction, resulting in specific downstream signaling and biological outcomes. Lysine methylation is a dynamic and fine-tuned process, deregulation of which often leads to human pathologies. In particular, the lysine methylome and its associated signaling network can be linked to carcinogenesis and cancer progression. Histone modifications and chromatin regulation is a major aspect of lysine methylation importance, but increasing evidence suggests that a high relevance and impact of non-histone lysine methylation signaling has emerged in recent years. In this review, we draw an updated picture of the current scientific knowledge regarding non-histone lysine methylation signaling and its implication in physiological and pathological processes. We aim to demonstrate the significance of lysine methylation as a major and yet underestimated posttranslational modification, and to raise the importance of this modification in both epigenetic and cellular signaling by focusing on the observed activities of SET- and 7ß-strandcontaining human lysine methyltransferases. Recent evidence suggests that what has been observed so far regarding lysine methylation's implication in human pathologies is only the tip of the iceberg. Therefore, the exploration of the "methylome network" raises the possibility to use these enzymes and their substrates as promising new therapeutic targets for the development of future epigenetic and methyllysine signaling cancer treatments.