RESUMO
Various approaches have been developed to target RNA and modulate its function with modes of action including binding and cleavage. Herein, we explored how small molecule binding is correlated with cleavage induced by heterobifunctional ribonuclease targeting chimeras (RiboTACs), where RNase L is recruited to cleave the bound RNA target, in a transcriptome-wide, unbiased fashion. Only a fraction of bound targets was cleaved by RNase L, induced by RiboTAC binding. Global analysis suggested that (i) cleaved targets generally form a region of stable structure that encompasses the small molecule binding site; (ii) cleaved targets have preferred RNase L cleavage sites nearby small molecule binding sites; (iii) RiboTACs facilitate a cellular interaction between cleaved targets and RNase L; and (iv) the expression level of the target influences the extent of cleavage observed. In one example, we converted a binder of LGALS1 (galectin-1) mRNA into a RiboTAC. In MDA-MB-231 cells, the binder had no effect on galectin-1 protein levels, while the RiboTAC cleaved LGALS1 mRNA, reduced galectin-1 protein abundance, and affected galectin-1-associated oncogenic cellular phenotypes. Using LGALS1, we further assessed additional factors including the length of the linker that tethers the two components of the RiboTAC, cellular uptake, and the RNase L-recruiting module on RiboTAC potency. Collectively, these studies may facilitate triangulation of factors to enable the design of RiboTACs.
Assuntos
Transcriptoma , Humanos , Linhagem Celular Tumoral , Endorribonucleases/metabolismo , Endorribonucleases/química , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Ribonucleases/metabolismo , Ribonucleases/químicaRESUMO
Ribonuclease targeting chimeras (RiboTACs) induce degradation of an RNA target by facilitating an interaction between an RNA and a ribonuclease (RNase). We describe the screening of a DNA-encoded library (DEL) to identify binders of monomeric RNase L to provide a compound that induced dimerization of RNase L, activating its ribonuclease activity. This compound was incorporated into the design of a next-generation RiboTAC that targeted the microRNA-21 (miR-21) precursor and alleviated a miR-21-associated cellular phenotype in triple-negative breast cancer cells. The RNA-binding module in the RiboTAC is Dovitinib, a known receptor tyrosine kinase (RTK) inhibitor, which was previously identified to bind miR-21 as an off-target. Conversion of Dovitinib into this RiboTAC reprograms the known drug to selectively affect the RNA target. This work demonstrates that DEL can be used to identify compounds that bind and recruit proteins with effector functions in heterobifunctional compounds.
Assuntos
MicroRNAs , Neoplasias de Mama Triplo Negativas , Humanos , Ribonucleases , DNARESUMO
Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs.
Assuntos
Antineoplásicos , Redutases-Desidrogenases de Cadeia Curta , Antineoplásicos/farmacologia , Estresse do Retículo Endoplasmático , Humanos , Lipídeos , Resposta a Proteínas não DobradasRESUMO
Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, which do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic-acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids.
RESUMO
Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors. Described here are easily synthesized isotopically labeled desthiobiotin azide (isoDTB) tags that shortened the chemoproteomic workflow and allowed an increased coverage of cysteines in bacterial systems. They were used to quantify 59 % of all cysteines in essential proteins in Staphylococcus aureus and enabled the discovery of 88 cysteines that showed high reactivity, which correlates with functional importance. Furthermore, 268 cysteines that are engaged by covalent ligands were identified. Inhibition of HMG-CoA synthase was verified and will allow addressing the bacterial mevalonate pathway through a new target. Overall, a broad map of the bacterial cysteinome was obtained, which will facilitate the development of antibiotics with novel modes-of-action.