A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2.

The pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further fragment expansion and drug development.

Regulation of MALAT1 triple helix stability and in vitro degradation by diphenylfurans.

Small molecule-based modulation of a triple helix in the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been proposed as an attractive avenue for cancer treatment and a model system for understanding small molecule:RNA recognition. To elucidate fundamental recognition principles and structure-function relationships, we designed and synthesized nine novel analogs of a diphenylfuran-based small molecule DPFp8, a previously identified lead binder of MALAT1. We investigated the role of recognition modalities in binding and in silico studies along with the relationship between affinity, stability and in vitro enzymatic degradation of the triple helix. Specifically, molecular docking studies identified patterns driving affinity and selectivity, including limited ligand flexibility, as observed by ligand preorganization and 3D shape complementarity for the binding pocket. The use of differential scanning fluorimetry allowed rapid evaluation of ligand-induced thermal stabilization of the triple helix, which correlated with decreased in vitro degradation of this structure by the RNase R exonuclease. The magnitude of stabilization was related to binding mode and selectivity between the triple helix and its precursor stem loop structure. Together, this work demonstrates the value of scaffold-based libraries in revealing recognition principles and of raising broadly applicable strategies, including functional assays, for small molecule-RNA targeting.

Structural basis of DNA duplex distortion induced by thiazole-containing hairpin polyamides.

This manuscript reports the molecular basis for double-stranded DNA (dsDNA) binding of hairpin polyamides incorporating a 5-alkyl thiazole (Nt) unit. Hairpin polyamides containing an N-terminal Nt unit induce higher melting stabilisation of target dsDNA sequences relative to an archetypical hairpin polyamide incorporating an N-terminal imidazole (Im) unit. However, modification of the N-terminus from Im to Nt-building blocks results in an increase in dsDNA binding affinity but lower G-selectivity. A general G-selectivity trend is observed for Nt-containing polyamide analogues. G-selectivity increases as the steric bulk in the Nt 5-position increases. Solution-based NMR structural studies reveal differences in the modulation of the target DNA duplex of Nt-containing hairpin polyamides relative to the Im-containing archetype. A structural hallmark of an Nt polyamide•dsDNA complex is a more significant degree of major groove compression of the target dsDNA sequence relative to the Im-containing hairpin polyamide.

Sequence-Selective Minor Groove Recognition of a DNA Duplex Containing Synthetic Genetic Components.

The structural basis of minor groove recognition of a DNA duplex containing synthetic genetic information by hairpin pyrrole-imidazole polyamides is described. Hairpin polyamides induce a higher melting stabilization of a DNA duplex containing the unnatural P·Z base-pair when an imidazole unit is aligned with a P nucleotide. An NMR structural study showed that the incorporation of two isolated P·Z pairs enlarges the minor groove and slightly narrows the major groove at the site of this synthetic genetic information, relative to a DNA duplex consisting entirely of Watson-Crick base-pairs. Pyrrole-imidazole polyamides bind to a P·Z-containing DNA duplex to form a stable complex, effectively mimicking a G·C pair. A structural hallmark of minor groove recognition of a P·Z pair by a polyamide is the reduced level of allosteric distortion induced by binding of a polyamide to a DNA duplex. Understanding the molecular determinants that influence minor groove recognition of DNA containing synthetic genetic components provides the basis to further develop unnatural base-pairs for synthetic biology applications.

Structural and Kinetic Profiling of Allosteric Modulation of Duplex DNA Induced by DNA-Binding Polyamide Analogues.

A combined structural and quantitative biophysical profile of the DNA binding affinity, kinetics and sequence-selectivity of hairpin polyamide analogues is described. DNA duplexes containing either target polyamide binding sites or mismatch sequences are immobilized on a microelectrode surface. Quantitation of the DNA binding profile of polyamides containing N-terminal 1-alkylimidazole (Im) units exhibit picomolar binding affinities for their target sequences, whereas 5-alkylthiazole (Nt) units are an order of magnitude lower (low nanomolar). Comparative NMR structural analyses of the polyamide series shows that the steric bulk distal to the DNA-binding face of the hairpin iPr-Nt polyamide plays an influential role in the allosteric modulation of the overall DNA duplex structure. This combined kinetic and structural study provides a foundation to develop next-generation hairpin designs where the DNA-binding profile of polyamides is reconciled with their physicochemical properties.

Inhibition of activated STAT5 in Bcr/Abl expressing leukemia cells with new pimozide derivatives.

STATs are transcription factors acting as intracellular signaling after stimulation with cytokines, growth factors and hormones. STAT5 is also constitutively active in many forms of cancers, including chronic myelogenous leukemia, acute lymphoblastic leukemia and Hodgkin's lymphoma. Recently, literature reported that the neuroleptic drug pimozide inhibits STAT5 phosphorylation inducing apoptosis in CML cells. We undertook an investigation from pimozide structure, obtaining simple derivatives with cytotoxic and STAT5-inhibitory activity, two of them markedly more potent than pimozide.

Specific protein-RNA interactions are mostly preserved in biomolecular condensates.

Many biomolecular condensates are enriched in and depend on RNAs and RNA binding proteins (RBPs). So far, only a few studies have addressed the characterization of the intermolecular interactions responsible for liquid-liquid phase separation (LLPS) and the impact of condensation on RBPs and RNAs. Here, we present an approach to study protein-RNA interactions inside biomolecular condensates by applying cross-linking of isotope labeled RNA and tandem mass spectrometry to phase-separating systems (LLPS-CLIR-MS). LLPS-CLIR-MS enables the characterization of intermolecular interactions present within biomolecular condensates at residue-specific resolution and allows a comparison with the same complexes in the dispersed phase. We observe that sequence-specific RBP-RNA interactions present in the dispersed phase are generally maintained inside condensates. In addition, LLPS-CLIR-MS identifies structural alterations at the protein-RNA interfaces, including additional unspecific contacts in the condensed phase. Our approach offers a procedure to derive structural information of protein-RNA complexes within biomolecular condensates that could be critical for integrative structural modeling of ribonucleoproteins (RNPs) in this form.

Differentiation and classification of RNA motifs using small molecule-based pattern recognition.

Understanding how to design small molecules that target coding and non-coding RNA has the potential to exponentially increase the number of therapeutically-relevant druggable targets, which are currently mostly proteins. However, there is limited information on the principles at the basis of RNA recognition. In this chapter, we describe a pattern-based technique that can be used for the simultaneous elucidation of RNA motifs and small molecule features for RNA selective recognition, termed Pattern Recognition of RNA by Small Molecules (PRRSM). We provide protocols for the computational design and synthetic preparation of an RNA training set as well as how to perform the assay in plate reader format. Furthermore, we provide details on how to perform and interpret the statistical analysis and indicate possible future extensions of the technique. By combining insights into characteristics of the small molecules and of the RNA that leads to differentiation, PRRSM promises to accelerate the elucidation of the determinants at the basis of RNA recognition.