HEX17(Neumifil): An intranasal respiratory biotherapeutic with broad-acting antiviral activity.

Broad-acting antiviral strategies to prevent respiratory tract infections are urgently required. Emerging or re-emerging viral diseases caused by new or genetic variants of viruses such as influenza viruses (IFVs), respiratory syncytial viruses (RSVs), human rhinoviruses (HRVs), parainfluenza viruses (PIVs) or coronaviruses (CoVs), pose a severe threat to human health, particularly in the very young or old, or in those with pre-existing respiratory conditions such as asthma or chronic obstructive pulmonary disease (COPD). Although vaccines remain a key component in controlling and preventing viral infections, they are unable to provide broad-spectrum protection against recurring seasonal infections or newly emerging threats. HEX17 (aka Neumifil), is a first-in-class protein-based antiviral prophylactic for respiratory viral infections. HEX17 consists of a hexavalent carbohydrate-binding module (CBM) with high affinity to sialic acids, which are typically present on terminating branches of glycans on viral cellular receptors. This allows HEX17 to block virus engagement of host receptors and inhibit infection of a wide range of viral pathogens and their variants with reduced risk of antiviral resistance. As described herein, HEX17 has demonstrated broad-spectrum efficacy against respiratory viral pathogens including IFV, RSV, CoV and HRV in multiple in vivo and in vitro studies. In addition, HEX17 can be easily administered via an intranasal spray and is currently undergoing clinical trials.

Structure and Function of the PiuA and PirA Siderophore-Drug Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii.

The outer membrane of Gram-negative bacteria presents an efficient barrier to the permeation of antimicrobial molecules. One strategy pursued to circumvent this obstacle is to hijack transport systems for essential nutrients, such as iron. BAL30072 and MC-1 are two monobactams conjugated to a dihydroxypyridone siderophore that are active against Pseudomonas aeruginosa and Acinetobacter baumannii Here, we investigated the mechanism of action of these molecules in A. baumannii We identified two novel TonB-dependent receptors, termed Ab-PiuA and Ab-PirA, that are required for the antimicrobial activity of both agents. Deletion of either piuA or pirA in A. baumannii resulted in 4- to 8-fold-decreased susceptibility, while their overexpression in the heterologous host P. aeruginosa increased susceptibility to the two siderophore-drug conjugates by 4- to 32-fold. The crystal structures of PiuA and PirA from A. baumannii and their orthologues from P. aeruginosa were determined. The structures revealed similar architectures; however, structural differences between PirA and PiuA point to potential differences between their cognate siderophore ligands. Spontaneous mutants, selected upon exposure to BAL30072, harbored frameshift mutations in either the ExbD3 or the TonB3 protein of A. baumannii, forming the cytoplasmic-membrane complex providing the energy for the siderophore translocation process. The results of this study provide insight for the rational design of novel siderophore-drug conjugates against problematic Gram-negative pathogens.

Interaction of kinase-interaction-motif protein tyrosine phosphatases with the mitogen-activated protein kinase ERK2.

The mitogen-activation protein kinase ERK2 is tightly regulated by multiple phosphatases, including those of the kinase interaction motif (KIM) PTP family (STEP, PTPSL and HePTP). Here, we use small angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC) to show that the ERK2:STEP complex is compact and that residues outside the canonical KIM motif of STEP contribute to ERK2 binding. Furthermore, we analyzed the interaction of PTPSL with ERK2 showing that residues outside of the canonical KIM motif also contribute to ERK2 binding. The integration of this work with previous studies provides a quantitative and structural map of how the members of a single family of regulators, the KIM-PTPs, differentially interact with their corresponding MAPKs, ERK2 and p38α.

The differential regulation of p38α by the neuronal kinase interaction motif protein tyrosine phosphatases, a detailed molecular study.

The MAP kinase p38α is essential for neuronal signaling. To better understand the molecular regulation of p38α we used atomistic and molecular techniques to determine the structural basis of p38α regulation by the two neuronal tyrosine phosphatases, PTPSL/PTPBR7 (PTPRR) and STEP (PTPN5). We show that, despite the fact that PTPSL and STEP belong to the same family of regulatory proteins, they interact with p38α differently and their distinct molecular interactions explain their different catalytic activities. Although the interaction of PTPSL with p38α is similar to that of the previously described p38α:HePTP (PTPN7) complex, STEP binds and regulates p38α in an unexpected manner. Using NMR and small-angle X-ray scattering data, we generated a model of the p38α:STEP complex and define molecular differences between its resting and active states. Together, these results provide insights into molecular regulation of p38α by key regulatory proteins.

Resting and active states of the ERK2:HePTP complex.

The MAP kinase ERK2 (ERK2, extracellular signal-regulated kinase 2) is regulated by numerous phosphatases that tightly control its activity. For example, the hematopoietic tyrosine phosphatase (HePTP) negatively regulates T cell activation in lymphocytes via ERK2 dephosphorylation. However, only very limited structural information is available for these biologically important complexes. Here, we use small-angle X-ray scattering combined with EROS ensemble refinement to characterize the structures of the resting and active states of ERK2:HePTP complexes. Our data show that the resting state ERK2:HePTP complex adopts a highly extended, dynamic conformation that becomes compact and ordered in the active state complex. This work experimentally demonstrates that these complexes undergo significant dynamic structural changes in solution and provides the first structural insight into an active state MAPK complex.

Structural basis of substrate recognition by hematopoietic tyrosine phosphatase.

Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPs as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.

Peptergents: peptide detergents that improve stability and functionality of a membrane protein, glycerol-3-phosphate dehydrogenase.

Toward enhancing in vitro membrane protein studies, we have utilized small self-assembling peptides with detergent properties ("peptergents") to extract and stabilize the integral membrane flavoenzyme, glycerol-3-phosphate dehydrogenase (GlpD), and the soluble redox flavoenzyme, NADH peroxidase (Npx). GlpD is a six transmembrane spanning redox enzyme that catalyzes the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate. Although detergents such as n-octyl-beta-D-glucpyranoside can efficiently solubilize the enzyme, GlpD is inactivated within days once reconstituted into detergent micelles. In contrast, peptergents can efficiently extract and solubilize GlpD from native Escherichia coli membrane and maintain its enzymatic activity up to 10 times longer than in traditional detergents. Intriguingly, peptergents also extended the activity of a soluble flavoenzyme, Npx, when used as an additive. Npx is a flavoenzyme that catalyzes the two-electron reduction of hydrogen peroxide to water using a cysteine-sulfenic acid as a secondary redox center. The lability of the peroxidase results from oxidation of the sulfenic acid to the sulfinic or sulfonic acid forms. Oxidation of the sulfenic acid, the secondary redox center, results in inactivation, and this reaction proceeds in vitro even in the presence of reducing agents. Although the exact mechanism by which peptergents influence solution stability of Npx remains to be determined, the positive effects may be due to antioxidant properties of the peptides. Peptide-based detergents can be beneficial for many applications and may be particularly useful for structural and functional studies of membrane proteins due to their propensity to enhance the formation of ordered supramolecular assemblies.