RESUMEN
Widespread adoption of mirror-image biological systems presents difficulties in accessing the requisite D-protein substrates. In particular, mirror-image phage display has the potential for high-throughput generation of biologically stable macrocyclic D-peptide binders with potentially unique recognition modes but is hindered by the individualized optimization required for D-protein chemical synthesis. We demonstrate a general mirror-image phage display pipeline that utilizes automated flow peptide synthesis to prepare D-proteins in a single run. With this approach, we prepare and characterize 12 D-proteins - almost one third of all reported D-proteins to date. With access to mirror-image protein targets, we describe the successful discovery of six macrocyclic D-peptide binders: three to the oncoprotein MDM2, and three to the E3 ubiquitin ligase CHIP. Reliable production of mirror-image proteins can unlock the full potential of D-peptide drug discovery and streamline the study of mirror-image biology more broadly.
Asunto(s)
Péptidos , Proteínas , Ligandos , Descubrimiento de DrogasRESUMEN
Peptide nucleic acids (PNAs) are potential antisense therapies for genetic, acquired, and viral diseases. Efficiently selecting candidate PNA sequences for synthesis and evaluation from a genome containing hundreds to thousands of options can be challenging. To facilitate this process, this work leverages machine learning (ML) algorithms and automated synthesis technology to predict PNA synthesis efficiency and guide rational PNA sequence design. The training data is collected from individual fluorenylmethyloxycarbonyl (Fmoc) deprotection reactions performed on a fully automated PNA synthesizer. The optimized ML model allows for 93% prediction accuracy and 0.97 Pearson's r. The predicted synthesis scores are validated to be correlated with the experimental high-performance liquid chromatography (HPLC) crude purities (correlation coefficient R2 = 0.95). Furthermore, a general applicability of ML is demonstrated through designing synthetically accessible antisense PNA sequences from 102 315 predicted candidates targeting exon 44 of the human dystrophin gene, SARS-CoV-2, HIV, as well as selected genes associated with cardiovascular diseases, type II diabetes, and various cancers. Collectively, ML provides an accurate prediction of PNA synthesis quality and serves as a useful computational tool for informing PNA sequence design.
Asunto(s)
COVID-19 , Diabetes Mellitus Tipo 2 , Ácidos Nucleicos de Péptidos , Humanos , Ácidos Nucleicos de Péptidos/genética , SARS-CoV-2/genética , Aprendizaje AutomáticoRESUMEN
Antisense peptide nucleic acids (PNAs) have yet to translate to the clinic because of poor cellular uptake, limited solubility, and rapid elimination. Cell-penetrating peptides (CPPs) covalently attached to PNAs may facilitate clinical development by improving uptake into cells. We report an efficient technology that utilizes a fully automated fast-flow instrument to manufacture CPP-conjugated PNAs (PPNAs) in a single shot. The machine is rapid, with each amide bond being formed in 10 s. Anti-IVS2-654 PPNA synthesized with this instrument presented threefold activity compared to transfected PNA in a splice-correction assay. We demonstrated the utility of this approach by chemically synthesizing eight anti-SARS-CoV-2 PPNAs in 1 day. A PPNA targeting the 5' untranslated region of SARS-CoV-2 genomic RNA reduced the viral titer by over 95% in a live virus infection assay (IC50 = 0.8 µM). Our technology can deliver PPNA candidates to further investigate their potential as antiviral agents.
RESUMEN
Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense phosphorodiamidate morpholino oligomers (PMOs) are promising candidates to fill such a role, but their challenging synthesis limits their widespread application. To rapidly prototype potential PMO drug candidates, we report a fully automated flow-based oligonucleotide synthesizer. Our optimized synthesis platform reduces coupling times by up to 22-fold compared to previously reported methods. We demonstrate the power of our automated technology with the synthesis of milligram quantities of three candidate therapeutic PMO sequences for an unserved class of Duchenne muscular dystrophy (DMD). To further test our platform, we synthesize a PMO that targets the genomic mRNA of SARS-CoV-2 and demonstrate its antiviral effects. This platform could find broad application not only in designing new SARS-CoV-2 and DMD antisense therapeutics, but also for rapid development of PMO candidates to treat new and emerging diseases.