The oriental hornet (Vespa orientalis) as a potential vector of honey bee's pathogens and a threat for public health in North-East Italy.

BACKGROUND: Oriental hornets are large predatory hymenoptera that occur in the southern part of Asia and the southeastern Mediterranean. Among many pests of bee colonies, Vespa orientalis was recorded to be one of the most destructive. OBJECTIVES: The aim of this study was to: (1) monitor the presence of pathogens carried by V. orientalis that could potentially threaten honey bees and public health; (2) describe the hornet's predatory behavior on honey bee colonies and (3) collect the medical history of a V. orientalis sting suffered by a 36-year-old woman. METHODS: Observations of V. orientalis predatory behavior and the catches of hornets for parasitological and microbiological examination, using molecular and bacteriological analyses, were carried out in three experimental apiaries, both in spring in order to capture the foundress queens and during the summer to capture the workers. Furthermore, the medical history and photographic documentation of a V. orientalis sting suffered by a 36-year-old woman have been collected. RESULTS: The results obtained highlight that V. orientalis is capable of causing serious damage to beekeeping by killing bees, putting under stress the honey bee colonies and by potentially spreading honey bee pathogens among apiaries. These hornets may also become a public health concern, since they are capable of inflicting multiple, painful stings on humans. CONCLUSIONS: Only the development of an Integrated Management Control Program will be able to contain the negative effects of anomalous population growth and the potentially negative impact on honey bees and public health of V. orientalis.

COVID-19 Genomic Surveillance in Bangui (Central African Republic) Reveals a Landscape of Circulating Variants Linked to Validated Antiviral Targets of SARS-CoV-2 Proteome.

Since its outbreak, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spread rapidly, causing the Coronavirus Disease 19 (COVID-19) pandemic. Even with the vaccines' administration, the virus continued to circulate due to inequal access to prevention and therapeutic measures in African countries. Information about COVID-19 in Africa has been limited and contradictory, and thus regional studies are important. On this premise, we conducted a genomic surveillance study about COVID-19 lineages circulating in Bangui, Central African Republic (CAR). We collected 2687 nasopharyngeal samples at four checkpoints in Bangui from 2 to 22 July 2021. Fifty-three samples tested positive for SARS-CoV-2, and viral genomes were sequenced to look for the presence of different viral strains. We performed phylogenetic analysis and described the lineage landscape of SARS-CoV-2 circulating in the CAR along 15 months of pandemics and in Africa during the study period, finding the Delta variant as the predominant Variant of Concern (VoC). The deduced aminoacidic sequences of structural and non-structural genes were determined and compared to reference and reported isolates from Africa. Despite the limited number of positive samples obtained, this study provides valuable information about COVID-19 evolution at the regional level and allows for a better understanding of SARS-CoV-2 circulation in the CAR.

Suramin inhibits SARS-CoV-2 nucleocapsid phosphoprotein genome packaging function.

The coronavirus disease 2019 (COVID-19) pandemic is fading, however its etiologic agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues posing - despite the availability of licensed vaccines - a global health threat, due to the potential emergence of vaccine-resistant SARS-CoV-2 variants. This makes the development of new drugs against COVID-19 a persistent urgency and sets as research priority the validation of novel therapeutic targets within the SARS-CoV-2 proteome. Among these, a promising one is the SARS-CoV-2 nucleocapsid (N) phosphoprotein, a major structural component of the virion with indispensable role in packaging the viral genome into a ribonucleoprotein (RNP) complex, which also contributes to SARS-CoV-2 innate immune evasion by inhibiting the host cell type-I interferon (IFN-I) response. By combining miniaturized differential scanning fluorimetry with microscale thermophoresis, we found that the 100-year-old drug Suramin interacts with SARS-CoV-2 N-terminal domain (NTD) and C-terminal domain (CTD), thereby inhibiting their single-stranded RNA (ssRNA) binding function with low-micromolar Kd and IC50 values. Molecular docking suggests that Suramin interacts with basic NTD cleft and CTD dimer interface groove, highlighting three potentially druggable ssRNA binding sites. Electron microscopy shows that Suramin inhibits the formation in vitro of RNP complex-like condensates by SARS-CoV-2 N with a synthetic ssRNA. In a dose-dependent manner, Suramin also reduced SARS-CoV-2-induced cytopathic effect on Vero E6 and Calu-3 cells, partially reverting the SARS-CoV-2 N-inhibited IFN-I production in 293T cells. Our findings indicate that Suramin inhibits SARS-CoV-2 replication by hampering viral genome packaging, thereby representing a starting model for design of new COVID-19 antivirals.

Ebolavirus VP40 redux. Or rather, redox.

The virion protein 40 (VP40) forms the viral matrix, mediates budding, and downregulates viral RNA synthesis in ebolaviruses. In this issue of Structure, Werner et al. present a structure of VP40 from Sudan ebolavirus with a previously unresolved disulfide bridge that enables regulation of VP40 functions via human thioredoxin.

Biophysical characterization of the cetacean morbillivirus haemagglutinin glycoprotein.

Cetacean morbillivirus (CeMV) is an enveloped, non-segmented, negative-stranded RNA virus that infects marine mammals, spreading across species and causing lethal disease outbreaks worldwide. Among the eight proteins encoded by the CeMV genome, the haemagglutinin (H) glycoprotein is responsible for the virus attachment to host cell receptors. CeMV H represents an attractive target for antiviral and diagnostic research, yet the elucidation of the molecular mechanisms underlying its role in infection and inter-species transmission was hampered thus far due to the unavailability of recombinant versions of the protein. Here we present the cloning, expression and purification of a recombinant CeMV H ectodomain (rH-ecto), providing an initial characterization of its biophysical and structural properties. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (PAGE) combined to Western blot analysis and periodic acid Schiff assay showed that CeMV rH-ecto is purifiable at homogeneity from insect cells as a secreted, soluble and glycosylated protein. Miniaturized differential scanning fluorimetry, Blue Native PAGE and size exclusion chromatography coupled to multiangle light scattering revealed that CeMV rH-ecto is globularly folded, thermally stable and exists in solution in the oligomeric states of dimer and multiple of dimers. Furthermore, negative stain electron microscopy single particle analysis allowed us to delineate a low-resolution molecular architecture of the CeMV rH-ecto dimer, which recapitulates native assemblies from other morbilliviral H proteins, such as those from measles virus and canine distemper virus. This set of experiments by orthogonal techniques validates the CeMV rH-ecto as an experimental model for future biochemical studies on its structure and functions.

Ebola and Marburg virus VP35 coiled-coil validated as antiviral target by tripartite split-GFP complementation.

Ebola virus (EBOV) and Marburg virus (MARV) are highly pathogenic viruses in humans, against which approved antivirals are lacking. During EBOV and MARV infection, coiled-coil mediated oligomerization is essential for the virion protein 35 (VP35) polymerase co-factor function and type I interferon antagonism, making VP35 coiled-coil an elective drug target. We established a tripartite split-green fluorescent protein (GFP) fluorescence complementation (FC) system based on recombinant GFP-tagged EBOV and MARV VP35, which probes VP35 coiled-coil assembly by monitoring fluorescence on E. coli colonies, or in vitro in 96/384-multiwell. Oligomerization-defective VP35 mutants showed that correct coiled-coil knobs-into-holes pairing within VP35 oligomer is pre-requisite for GFP tags and GFP detector to reconstitute fluorescing full-length GFP. The method was validated by screening a small compound library, which identified Myricetin and 4,5,6,7-Tetrabromobenzotriazole as inhibitors of EBOV and MARV VP35 oligomerization-dependent FC with low-micromolar IC50 values. These findings substantiate the VP35 coiled-coil value as antiviral target.

Brucella ceti and Brucella pinnipedialis genome characterization unveils genetic features that highlight their zoonotic potential.

The Gram-negative bacteria Brucella ceti and Brucella pinnipedialis circulate in marine environments primarily infecting marine mammals, where they cause an often-fatal disease named brucellosis. The increase of brucellosis among several species of cetaceans and pinnipeds, together with the report of sporadic human infections, raises concerns about the zoonotic potential of these pathogens on a large scale and may pose a threat to coastal communities worldwide. Therefore, the characterization of the B. ceti and B. pinnipedialis genetic features is a priority to better understand the pathological factors that may impact global health. Moreover, an in-depth functional analysis of the B. ceti and B. pinnipedialis genome in the context of virulence and pathogenesis was not undertaken so far. Within this picture, here we present the comparative whole-genome characterization of all B. ceti and B. pinnipedialis genomes available in public resources, uncovering a collection of genetic tools possessed by these aquatic bacterial species compared to their zoonotic terrestrial relatives. We show that B. ceti and B. pinnipedialis genomes display a wide host-range infection capability and a polyphyletic phylogeny within the genus, showing a genomic structure that fits the canonical definition of closeness. Functional genome annotation led to identifying genes related to several pathways involved in mechanisms of infection, others conferring pan-susceptibility to antimicrobials and a set of virulence genes that highlight the similarity of B. ceti and B. pinnipedialis genotypes to those of Brucella spp. displaying human-infecting phenotypes.

Cynarin blocks Ebola virus replication by counteracting VP35 inhibition of interferon-beta production.

Ebola virus (EBOV) is one of the deadliest infective agents whose lethality is linked to the ability to efficiently bypass the host's innate antiviral response. EBOV multifunctional protein VP35 plays a major role in viral replication both as polymerase cofactor and interferon (IFN) antagonist. By hiding the non-self 5'-ppp dsRNA from the cellular receptor RIG-I, VP35 prevents its activation and inhibits IFN-ß production. Blocking VP35-dsRNA interaction and IFN-ß suppression is a validated drug target. We screened a library of natural extracts and found that cynarin inhibits dsRNA-VP35 binding with an IC50 value of 8.5 µM. It reverts the EBOV VP35 inhibition of IFN-ß production, while it does not induce IFN production by itself. Docking experiments suggest that the molecule can bind on the end-capping pocket of VP35 C-terminal Interferon Inhibitory domain (IID), and differential scanning fluorimetry confirmed that cynarin interacts with VP35-IID with a KD of 12 µM. Cynarin was further tested in an EBOV minigenome assay but did not inhibit VP35 polymerase cofactor activity. When evaluated during challenge of IFN-susceptible A549 cells with EBOV isolate derived from the 2014 West African outbreak, cynarin was able to inhibit viral replication with an EC50 value of 9.1 µM, showing no significant cytotoxicity. Our findings show that cynarin blocks EBOV replication by acting directly on VP35 and subverting its IFN antagonism function but not cofactor function, and as such identify the first EBOV inhibitor with this mode of action.

Molecular signatures in cetacean morbillivirus and host species proteomes: Unveiling the evolutionary dynamics of an enigmatic pathogen?

Cetacean morbillivirus (CeMV) infects marine mammals often causing a fatal respiratory and neurological disease. Recently, CeMV has expanded its geographic and host species range, with cases being reported worldwide among dolphins, whales, seals, and other aquatic mammalian species, and therefore has emerged as the most threatening nonanthropogenic factor affecting marine mammal's health and conservation. Extensive research efforts have aimed to understand CeMV epidemiology and ecology, however, the molecular mechanisms underlying its transmission and pathogenesis are still poorly understood. In particular, the field suffers from a knowledge gap on the structural and functional properties of CeMV proteins and their host interactors. Nevertheless, the body of scientific literature produced in recent years has inaugurated new investigational trends, driving future directions in CeMV molecular research. In this mini-review, the most recent literature has been summarized in the context of such research trends, and categorized into four priority research topics, such as (1) the interaction between CeMV glycoprotein and its host cell receptors across several species; (2) the CeMV molecular determinants responsible for different disease phenotype; (3) the host molecular determinants responsible for differential susceptibility to CeMV infection; (4) the CeMV molecular determinants responsible for difference virulence among circulating CeMV strains. Arguably, these are the most urgent topics that need to be investigated and that most promisingly will help to shed light on the details of CeMV evolutionary dynamics in the immediate future.

Cryo-EM structure of the cetacean morbillivirus nucleoprotein-RNA complex.

Cetacean morbillivirus (CeMV) is an emerging and highly infectious paramyxovirus that causes outbreaks in cetaceans and occasionally in pinnipeds, representing a major threat to biodiversity and conservation of endangered marine mammal populations in both hemispheres. As for all non-segmented, negative-sense, single-stranded RNA (ssRNA) viruses, the morbilliviral genome is enwrapped by thousands of nucleoprotein (N) protomers. Each bound to six ribonucleotides, N protomers assemble to form a helical ribonucleoprotein (RNP) complex that serves as scaffold for nucleocapsid formation and as template for viral replication and transcription. While the molecular details on RNP complexes elucidated in human measles virus (MeV) served as paradigm model for these processes in all members of the Morbillivirus genus, no structural information has been obtained from other morbilliviruses, nor has any CeMV structure been solved so far. We report the structure of the CeMV RNP complex, reconstituted in vitro upon binding of recombinant CeMV N to poly-adenine ssRNA hexamers and solved to 4.0 Å resolution by cryo-electron microscopy. In spite of the amino acid sequence similarity and consequently similar folding of the N protomer, the CeMV RNP complex exhibits different helical parameters as compared to previously reported MeV orthologs. The CeMV structure reveals exclusive interactions leading to more extensive protomer-RNA and protomer-protomer interfaces. We identified twelve residues, among those varying between CeMV strains, as putatively important for the stabilization of the RNP complex, which highlights the need to study the potential of CeMV N mutations that modulate nucleocapsid assembly to also affect viral phenotype and host adaptation.

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-ß pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.

Lost in deletion: The enigmatic ORF8 protein of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome contains nine open reading frames (ORFs) that encode for accessory proteins which, although dispensable for viral replication, are important for the modulation of the host infected cell metabolism and innate immunity evasion. Among those, the ORF8 gene encodes for the homonymous multifunctional, highly immunogenic, immunoglobulin-like protein that was recently found to inhibit presentation of viral antigens by class I major histocompatibility complex, suppress the type I interferon antiviral response and interact with host factors involved in pulmonary inflammation and fibrogenesis. Moreover, the ORF8 is a hypervariable gene rapidly evolving among SARS-related coronaviruses, with a tendency to recombine and undergo deletions that are deemed to facilitate the virus adaptation to the human host. Intriguingly, SARS-CoV-2 variants isolated in the beginning of the coronavirus disease 2019 (Covid-19) pandemic that were deleted of the ORF8 gene have been associated to milder symptoms and better disease outcome. This minireview summarizes the current knowledge on the SARS-CoV-2 ORF8 protein in perspective to its potential as antiviral target and with special emphasis on the biochemical, biophysical and structural aspects of its molecular biology.

Trans-synaptic assemblies link synaptic vesicles and neuroreceptors.

Synaptic transmission is characterized by fast, tightly coupled processes and complex signaling pathways that require a precise protein organization, such as the previously reported nanodomain colocalization of pre- and postsynaptic proteins. Here, we used cryo-electron tomography to visualize synaptic complexes together with their native environment comprising interacting proteins and lipids on a 2- to 4-nm scale. Using template-free detection and classification, we showed that tripartite trans-synaptic assemblies (subcolumns) link synaptic vesicles to postsynaptic receptors and established that a particular displacement between directly interacting complexes characterizes subcolumns. Furthermore, we obtained de novo average structures of ionotropic glutamate receptors in their physiological composition, embedded in plasma membrane. These data support the hypothesis that synaptic function is carried by precisely organized trans-synaptic units. It provides a framework for further exploration of synaptic and other large molecular assemblies that link different cells or cellular regions and may require weak or transient interactions to exert their function.

High-resolution structure and biophysical characterization of the nucleocapsid phosphoprotein dimerization domain from the Covid-19 severe acute respiratory syndrome coronavirus 2.

Unprecedented by number of casualties and socio-economic burden occurring worldwide, the coronavirus disease 2019 (Covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the worst health crisis of this century. In order to develop adequate countermeasures against Covid-19, identification and structural characterization of suitable antiviral targets within the SARS-CoV-2 protein repertoire is urgently needed. The nucleocapsid phosphoprotein (N) is a multifunctional and highly immunogenic determinant of virulence and pathogenicity, whose main functions consist in oligomerizing and packaging the single-stranded RNA (ssRNA) viral genome. Here we report the structural and biophysical characterization of the SARS-CoV-2 N C-terminal domain (CTD), on which both N homo-oligomerization and ssRNA binding depend. Crystal structures solved at 1.44 Å and 1.36 Å resolution describe a rhombus-shape N CTD dimer, which stably exists in solution as validated by size-exclusion chromatography coupled to multi-angle light scattering and analytical ultracentrifugation. Differential scanning fluorimetry revealed moderate thermal stability and a tendency towards conformational change. Microscale thermophoresis demonstrated binding to a 7-bp SARS-CoV-2 genomic ssRNA fragment at micromolar affinity. Furthermore, a low-resolution preliminary model of the full-length SARS-CoV N in complex with ssRNA, obtained by cryo-electron microscopy, provides an initial understanding of self-associating and RNA binding functions exerted by the SARS-CoV-2 N.

Structures of Ebola and Reston Virus VP35 Oligomerization Domains and Comparative Biophysical Characterization in All Ebolavirus Species.

The multifunctional virion protein 35 (VP35) of ebolaviruses is a critical determinant of virulence and pathogenesis indispensable for viral replication and host innate immune evasion. Essential for VP35 function is homo-oligomerization via a coiled-coil motif. Here we report crystal structures of VP35 oligomerization domains from the prototypic Ebola virus (EBOV) and the non-pathogenic Reston virus (RESTV), together with a comparative biophysical characterization of the domains from all known species of the Ebolavirus genus. EBOV and RESTV VP35 oligomerization domains form bipartite parallel helix bundles with a canonical coiled coil in the N-terminal half and increased plasticity in the highly conserved C-terminal half. The domain assembles into trimers and tetramers in EBOV, whereas it exclusively forms tetramers in all other ebolavirus species. Substitution of coiled-coil leucine residues critical for immune antagonism leads to aberrant oligomerization. A conserved arginine involved in inter-chain salt bridges stabilizes the VP35 oligomerization domain and modulates between coiled-coil oligomeric states.

Proteomics Analysis of Thermoplasma Quinone Droplets.

A novel type of lipid droplet/lipoprotein (LD/LP) particle from Thermoplasma acidophilum has been identified recently, and based on biochemical evidences, it was named Thermoplasma Quinone Droplet (TaQD). The major components of TaQDs are menaquinones, and to some extent polar lipids, and the 153 amino acid long Ta0547 vitellogenin-N domain protein. In this paper, the aim is to identify TaQD proteome components with 1D-SDS-PAGE/LC-MS/MS and cross reference them with Edman degradation. TaQD samples isolated with three different purification methods-column chromatography, immunoprecipitation, and LD ultracentrifugation-are analyzed. Proteins Ta0093, Ta0182, Ta0337, Ta0437, Ta0438, Ta0547, and Ta1223a are identified as constituents of the TaQD proteome. The majority of these proteins is uncharacterized and has low molecular weight, and none of them is predicted to take part in lipid metabolism. Bioinformatics analyses does not predict any interaction between these proteins, however, there are indications of interactions with proteins taking part in lipid metabolism. Whether if TaQDs provide platform for lipid metabolism and the interactions between TaQD proteins and lipid metabolism proteins occur in the reality remain for further studies.

Identification of Myricetin as an Ebola Virus VP35-Double-Stranded RNA Interaction Inhibitor through a Novel Fluorescence-Based Assay.

Ebola virus (EBOV) is a filovirus that causes a severe and rapidly progressing hemorrhagic syndrome; a recent epidemic illustrated the urgent need for novel therapeutic agents because no drugs have been approved for treatment of Ebola virus. A key contribution to the high lethality observed during EBOV outbreaks comes from viral evasion of the host antiviral innate immune response in which viral protein VP35 plays a crucial role, blocking interferon type I production, first by masking the viral double-stranded RNA (dsRNA) and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of the interaction of VP35 with the viral dsRNA, counteracting the VP35 viral innate immune evasion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification and a novel and robust fluorescence-based rVP35-RNA interaction assay ( Z' factor of 0.69). Taking advantage of such newly established methods, we screened a small library of Sardinian natural extracts, identifying Limonium morisianum as the most potent inhibitor extract. A bioguided fractionation led to the identification of myricetin as the component that can inhibit rVP35-dsRNA interaction with an IC50 value of 2.7 µM. Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent inhibitors of VP35-dsRNA interaction.

Insights into the homo-oligomerization properties of N-terminal coiled-coil domain of Ebola virus VP35 protein.

The multifunctional Ebola virus (EBOV) VP35 protein is a key determinant of virulence. VP35 is essential for EBOV replication, is a component of the viral RNA polymerase and participates in nucleocapsid formation. Furthermore, VP35 contributes to EBOV evasion of the host innate immune response by suppressing RNA silencing and blocking RIG-I like receptors' pathways that lead to type I interferon (IFN) production. VP35 homo-oligomerization has been reported to be critical for its replicative function and to increase its IFN-antagonism properties. Moreover, homo-oligomerization is mediated by a predicted coiled-coil (CC) domain located within its N-terminal region. Here we report the homo-oligomerization profile of full-length recombinant EBOV VP35 (rVP35) assessed by size-exclusion chromatography and native polyacrylamide gel electrophoresis. Based on our biochemical results and in agreement with previous experimental observations, we have built an in silico 3D model of the so-far structurally unsolved EBOV VP35 CC domain and performed self-assembly homo-oligomerization simulations by means of molecular dynamics. Our model advances the understanding of how VP35 may associate in different homo-oligomeric species, a crucial process for EBOV replication and pathogenicity.

Enhancing recombinant protein solubility with ubiquitin-like small archeal modifying protein fusion partners.

A variety of protein expression tags with different biochemical properties has been used to enhance the yield and solubility of recombinant proteins. Ubiquitin, SUMO (small ubiquitin-like modifier) and prokaryotic ubiquitin like MoaD (molybdopterin synthase, small subunit) fusion tags are getting more popular because of their small size. In this paper we report on the use of ubiquitin-like small archaeal modifier proteins (SAMPs) as fusion tags since they proved to increase expression yield, stability and solubility in our experiments. Equally important, they did not co-purify with proteins of the expression host and there was information that their specific JAB1/MPN/Mov34 metalloenzyme (JAMM) protease can recognize the C-terminal VSGG sequence when SAMPs fused, either branched or linearly to target proteins, and cleave it specifically. SAMPs and JAMM proteases from Haloferax volcanii, Thermoplasma acidophilum, Methanococcoides burtonii and Nitrosopumilus maritimus were selected, cloned, expressed heterologously in Escherichia coli and tested as fusion tags and cleaving proteases, respectively. Investigated SAMPs enhanced protein expression and solubility on a wide scale. T. acidophilum SAMPs Ta0895 and Ta01019 were the best performing tags and their effect was comparable to the widely used maltose binding protein (MBP) and N utilization substance protein A (NusA) tags. Moreover, H. volcanii SAMP Hvo_2619 contribution was mediocre, whereas M. burtonii Mbur_1415 could not be expressed. Out of four investigated JAMM proteases, only Hvo_2505 could cleave fusion tags. Interestingly, it was found active not only on its own partner substrate Hvo_2619, but it also cleaved off Ta0895.

6-(1-Benzyl-1H-pyrrol-2-yl)-2,4-dioxo-5-hexenoic acids as dual inhibitors of recombinant HIV-1 integrase and ribonuclease H, synthesized by a parallel synthesis approach.

The increasing efficiency of HAART has helped to transform HIV/AIDS into a chronic disease. Still, resistance and drug-drug interactions warrant the development of new anti-HIV agents. We previously discovered hit 6, active against HIV-1 replication and targeting RNase H in vitro. Because of its diketo-acid moiety, we speculated that this chemotype could serve to develop dual inhibitors of both RNase H and integrase. Here, we describe a new series of 1-benzyl-pyrrolyl diketohexenoic derivatives, 7a-y and 8a-y, synthesized following a parallel solution-phase approach. Those 50 analogues have been tested on recombinant enzymes (RNase H and integrase) and in cell-based assays. Approximately half (22) exibited inhibition of HIV replication. Compounds 7b, 7u, and 8g were the most active against the RNase H activity of reverse-transcriptase, with IC50 values of 3, 3, and 2.5 µM, respectively. Compound 8g was also the most potent integrase inhibitor with an IC50 value of 26 nM.