Through screening by NMR spectroscopy, we discovered a novel scaffold (DPQ: 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine) that binds specifically to the influenza A virus promoter RNA. The solution structure of the RNA-DPQ complex reported here demonstrates that the internal loop is the binding site of DPQ. The scaffold has antiviral activity against influenza viruses.

The emergence of drug-resistant strains of influenza virus, makes exploring new classes of inhibitors that target universally conserved viral targets a highly important goal. The influenza A viral genome is made up of 8 single-stranded RNA negative segments. The RNA promoter, consisting of the conserved sequences at the 3′ and 5′ end of each RNA genomic segment, is universally conserved among influenza A virus strains and in all segments. Previously we reported on the identification and NMR structure of DPQ (6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine) (compound 1) in complex with the RNA promoter. Here we report on additional screening and SAR studies with compound 1, including ex vivo anti-influenza activity assays, resulted in improved cellular activity against influenza A virus in the micromolar range.

ObjectiveWe aimed to understand the role of the tyrosine phosphatase PTPN14—which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol—in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).MethodsGene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis.ResultsRA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFβ-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP—but not of a non-YAP-interacting PTPN14 mutant—enhanced SMAD reporter activity. YAP promoted TGFβ-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity.ConclusionIn RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour.

Recently we described a novel approach, named HTS by NMR that allows the identification, from large combinatorial peptide libraries, of potent and selective peptide mimetics against a given target. Here we deployed the HTS by NMR approach for the design of novel peptoid sequences targeting the amino terminal domain of the Yersinia outer protein H (YopH-NT). We aimed at disrupting the protein-protein interactions between YopH-NT and its cellular substrates, with the goal of inhibiting indirectly YopH enzymatic function. These studies resulted in a novel agent of sequence Ac-F-pY-cPG-D-P-NH2 (pY = phosphotyrosine; cPG = cyclopentyl glycine) with a Kd value against YopH-NT of 310 nM. We demonstrated that such pharmacological inhibitor of YopH-NT resulted in the inhibition of the dephosphorylation of a cellular substrate by full length YopH. Hence, potentially this agent represents a valuable stepping stone for the development of novel therapeutics against Yersinia infections. The data reported further demonstrate the utility of the HTS by NMR approach in deriving novel peptide-mimetics targeting protein-protein interactions.