Revisiting VH1 phosphatase at the time of monkeypox: back to the spotlight.

Vaccinia virus is a poxvirus that has been successfully leveraged to develop vaccines for smallpox, which is caused by the closely related Variola virus. Smallpox has been declared as 'eradicated' by the WHO in 1980; however, it still poses a potential bioterrorism threat. More recently, the spreading of monkeypox (MPox) in non-endemic countries has further highlighted the importance of continuing the exploration for druggable targets for poxvirus infections. The vaccinia H1 (VH1) phosphatase is the first reported dual specificity phosphatase (DUSP) able to hydrolyze both phosphotyrosine and phosphoserine/phosphotheonine residues. VH1 is a 20 kDa protein that forms a stable dimer and can dephosphorylate both viral and cellular substrates to regulate the viral replication cycle and host immune response. VH1 dimers adopt a domain swap mechanism with the first 20 amino acids of each monomer involved in dense electrostatic interaction and salt bridge formations while hydrophobic interactions between the N-terminal and C-terminal helices further stabilize the dimer. VH1 appears to be an ideal candidate for discovery of novel anti-poxvirus agents because it is highly conserved within the poxviridae family and is a virulence factor, yet it displays significant divergence in sequence and dimerization mechanism from its human closest ortholog vaccinia H1-related (VHR) phosphatase, encoded by the DUSP3 gene. As the dimeric quaternary structure of VH1 is essential for its phosphatase activity, strategies leading to disruption of the dimer structure might aid in VH1 inhibitor development.

PTPN14 phosphatase and YAP promote TGFß signalling in rheumatoid synoviocytes.

OBJECTIVE: We 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). METHODS: Gene/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. RESULTS: RA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFß-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 TGFß-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. CONCLUSION: In 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.

High-Throughput Screening (HTS) by NMR Guided Identification of Novel Agents Targeting the Protein Docking Domain of YopH.

Recently we described a novel approach, named high-throughput screening (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 N-terminal domain of Yersinia outer protein H (YopH-NT), a bacterial toxin essential for the virulence of Yersinia pestis. 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 a pharmacological inhibitor of YopH-NT results in the inhibition of the dephosphorylation by full-length YopH of a cellular substrate. 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.

Targeting Influenza A Virus RNA Promoter.

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 eight 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.

A novel small-molecule binds to the influenza A virus RNA promoter and inhibits viral replication.

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 RNA promoter. The solution structure of the RNA-DPQ complex reported here demonstrates that the internal loop is the binding site of DPQ. The scaffold exhibits antiviral activity against influenza viruses.

Identification of small molecules that interfere with H1N1 influenza A viral replication.

Successful replication of the influenza A virus requires both viral proteins and host cellular factors. In this study we used a cellular assay to screen for small molecules capable of interfering with any of such necessary viral or cellular components. We used an established reporter assay to assess influenza viral replication by monitoring the activity of co-expressed luciferase. We screened a diverse chemical compound library, resulting in the identification of compound 7, which inhibits a novel yet elusive target. Quantitative real-time PCR studies confirmed the dose-dependent inhibitory activity of compound 7 in a viral replication assay. Furthermore, we showed that compound 7 is effective in rescuing high-dose influenza infection in an in vivo mouse model. As oseltamivir-resistant influenza strains emerge, compound 7 could be further investigated as a new and potentially suitable scaffold for the development of anti-influenza agents that act on novel targets.