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We designed novel pre-drug compounds that transform into an active form that covalently modifies particular His residue in the active site, a difficult task to achieve, and applied to carbonic anhydrase (CAIX), a transmembrane protein, highly overexpressed in hypoxic solid tumors, important for cancer cell survival and proliferation because it acidifies tumor microenvironment helping invasion and metastases processes. The designed compounds have several functionalities: (1) primary sulfonamide group recognizing carbonic anhydrases (CA), (2) high-affinity moieties specifically recognizing CAIX among all CA isozymes, and (3) forming a covalent bond with the His64 residue. Such targeted covalent compounds possess both high initial affinity and selectivity for the disease target protein followed by complete irreversible inactivation of the protein via covalent modification. Our designed prodrug candidates bearing moderately active pre-vinylsulfone esters or weakly active carbamates optimized for mild covalent modification activity to avoid toxic non-specific modifications and selectively target CAIX. The lead inhibitors reached 2 pM affinity, the highest among known CAIX inhibitors. The strategy could be used for any disease drug target protein bearing a His residue in the vicinity of the active site., Competing Interests: AV, DB, AZ, VD, DM has a patent application on CA inhibitors pending, JL, AK, AR, EM, ZT, AK, AK, MG, AM, VJ, HS, KJ, MV, KT, SG, FM, JM No competing interests declared, (© 2024, Vaškevičius et al.)

Declared as a Public Health Emergency in 2016 by the World Health Organization (WHO), the Zika virus (ZIKV) continues to cause outbreaks that are linked to increased neurological complications. Transmitted mainly by Aedes mosquitoes, the virus is spread mostly amongst several tropical regions with the potential of territorial expansion due to environmental and ecological changes. The ZIKV envelope protein's domain III, crucial for vaccine development due to its role in receptor binding and neutralizing antibody targeting, was integrated into sterically optimized AP205 VLPs to create an EDIII-based VLP vaccine. To increase the potential size of domains that can be accommodated by AP205, two AP205 monomers were fused into a dimer, resulting in 90 rather than 180 N-/C- termini amenable for fusion. EDIII displayed on AP205 VLPs has several immunological advantages, like a repetitive surface, a size of 20-200 nm (another PASP), and packaged bacterial RNA as adjuvants (a natural toll-like receptor 7/8 ligand). In this study, we evaluated a novel vaccine candidate for safety and immunogenicity in mice, demonstrating its ability to induce high-affinity, ZIKV-neutralizing antibodies without significant disease-enhancing properties. Due to the close genetical and structural characteristics, the same mosquito vectors, and the same ecological niche of the dengue virus and Zika virus, a vaccine covering all four Dengue viruses (DENV) serotypes as well as ZIKV would be of significant interest. We co-formulated the ZIKV vaccine with recently developed DENV vaccines based on the same AP205 VLP platform and tested the vaccine mix in a murine model. This combinatory vaccine effectively induced a strong humoral immune response and neutralized all five targeted viruses after two doses, with no significant antibody-dependent enhancement (ADE) observed. Overall, these findings highlight the potential of the AP205 VLP-based combinatory vaccine as a promising approach for providing broad protection against DENV and ZIKV infections. Further investigations and preclinical studies are required to advance this vaccine candidate toward potential use in human populations.

Over the past few decades, dengue fever has emerged as a significant global health threat, affecting tropical and moderate climate regions. Current vaccines have practical limitations, there is a strong need for safer, more effective options. This study introduces novel vaccine candidates covering all four dengue virus (DENV) serotypes using virus-like particles (VLPs), a proven vaccine platform. The dengue virus envelope protein domain III (EDIII), the primary target of DENV-neutralizing antibodies, was either genetically fused or chemically coupled to bacteriophage-derived AP205-VLPs. To facilitate the incorporation of the large EDIII domain, AP205 monomers were dimerized, resulting in sterically optimized VLPs with 90 N- and C-termini. These vaccines induced high-affinity/avidity antibody titers in mice, and confirmed their protective potential by neutralizing different DENV serotypes in vitro. Administration of a tetravalent vaccine induced high neutralizing titers against all four serotypes without producing enhancing antibodies, at least not against DENV2. In conclusion, the vaccine candidates, especially when administered in a combined fashion, exhibit intriguing properties for potential use in the field, and exploring the possibility of conducting a preclinical challenge model to verify protection would be a logical next step.

Carbonic anhydrase (CA) was among the first proteins whose X-ray crystal structure was solved to atomic resolution. CA proteins have essentially the same fold and similar active centers that differ in only several amino acids. Primary sulfonamides are well defined, strong and specific binders of CA. However, minor variations in chemical structure can significantly alter their binding properties. Over 1000 sulfonamides have been designed, synthesized and evaluated to understand the correlations between the structure and thermodynamics of their binding to the human CA isozyme family. Compound binding was determined by several binding assays: fluorescence-based thermal shift assay, stopped-flow enzyme activity inhibition assay, isothermal titration calorimetry and competition assay for enzyme expressed on cancer cell surfaces. All assays have advantages and limitations but are necessary for deeper characterization of these protein-ligand interactions. Here, the concept and importance of intrinsic binding thermodynamics is emphasized and the role of structure-thermodynamics correlations for the novel inhibitors of CA IX is discussed - an isozyme that is overexpressed in solid hypoxic tumors, and thus these inhibitors may serve as anticancer drugs. The abundant structural and thermodynamic data are assembled into the Protein-Ligand Binding Database to understand general protein-ligand recognition principles that could be used in drug discovery., (open access.)

Lyme disease is the most prevalent vector-borne infectious disease in Europe and the USA. Borrelia burgdorferi, as the causative agent of Lyme disease, is transmitted to the mammalian host during the tick blood meal. To adapt to the different encountered environments, Borrelia has adjusted the expression pattern of various, mostly outer surface proteins. The function of most B. burgdorferi outer surface proteins remains unknown. We determined the crystal structure of a previously uncharacterized B. burgdorferi outer surface protein BBK01, known to belong to the paralogous gene family 12 (PFam12) as one of its five members. PFam12 members are shown to be upregulated as the tick starts its blood meal. Structural analysis of BBK01 revealed similarity to the coiled coil domain of structural maintenance of chromosomes (SMC) protein family members, while functional studies indicated that all PFam12 members are non-specific DNA-binding proteins. The residues involved in DNA binding were identified and probed by site-directed mutagenesis. The combination of SMC-like proteins being attached to the outer membrane and exposed to the environment or located in the periplasm, as observed in the case of PFam12 members, and displaying the ability to bind DNA, represents a unique feature previously not observed in bacteria., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Brangulis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lyme disease, or also known as Lyme borreliosis, is caused by the spirochetes belonging to the Borrelia burgdorferi sensu lato complex, which can enter the human body following the bite of an infected tick. Many membrane lipid-bound proteins, also known as lipoproteins, are located on the surface of B. burgdorferi sensu lato and play a crucial role in the spirochete to interact with its environment, whether in ticks or mammals. Since the spirochete needs to perform various tasks, such as resisting the host's immune system or spreading throughout the organism, it is not surprising that numerous surface proteins have been found to be essential for B. burgdorferi sensu lato complex bacteria in causing Lyme disease. In this study, we have determined (at 2.4 Å resolution) and characterized the 3D structure of BB0158, one of the few chromosomally encoded outer surface proteins from B. burgdorferi sensu stricto. BB0158 belongs to the paralogous gene family 44 (PFam44), consisting of four other members (BB0159, BBA04, BBE09 and BBK52). The characterization of BB0158, which appears to form a domain-swapped dimer, in conjunction with the characterization of the corresponding PFam44 members, certainly contribute to our understanding of B. burgdorferi sensu stricto proteins., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest in this study., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). Nuclear magnetic resonance studies show a Pro-VPg complex interaction and VPg tertiary structure; however, information regarding structural changes of the Pro-VPg complex during interaction is lacking. Here, we solved a full Pro-VPg 3D structure of ryegrass mottle virus (RGMoV) that demonstrates the structural changes in three different conformations due to VPg interaction with Pro. We identified a unique site of VPg interaction with Pro that was not observed in other sobemoviruses, and observed different conformations of the Pro β2 barrel. This is the first report of a full plant Pro crystal structure with its VPg cofactor. We also confirmed the existence of an unusual previously unmapped cleavage site for sobemovirus Pro in the transmembrane domain: E/A. We demonstrated that RGMoV Pro in cis activity is not regulated by VPg and that in trans , VPg can also mediate Pro in free form. Additionally, we observed Ca 2+ and Zn 2+ inhibitory effects on the Pro cleavage activity.

Endolysins are bacteriophage-encoded peptidoglycan-degrading enzymes with potential applications for treatment of multidrug-resistant bacterial infections. Hafnia phage Enc34 encodes an unusual endolysin with an N-terminal enzymatically active domain and a C-terminal transmembrane domain. The catalytic domain of the endolysin belongs to the conserved protein family PHA02564 which has no recognizable sequence similarity to other known endolysin types. Turbidity reduction assays indicate that the Enc34 enzyme is active against peptidoglycan from a variety of Gram-negative bacteria including the opportunistic pathogen Pseudomonas aeruginosa PAO1. The crystal structure of the catalytic domain of the Enc34 endolysin shows a distinctive all-helical architecture that distantly resembles the α-lobe of the lysozyme fold. Conserved catalytically important residues suggest a shared evolutionary history between the Enc34 endolysin and GH73 and GH23 family glycoside hydrolases and propose a molecular signature for substrate cleavage for a large group of peptidoglycan-degrading enzymes., (© 2022. The Author(s).)

Among the twelve catalytically active carbonic anhydrase isozymes present in the human body, the CAIX is highly overexpressed in various solid tumors. The enzyme acidifies the tumor microenvironment enabling invasion and metastatic processes. Therefore, many attempts have been made to design chemical compounds that would exhibit high affinity and selective binding to CAIX over the remaining eleven catalytically active CA isozymes to limit undesired side effects. It has been postulated that such drugs may have anticancer properties and could be used in tumor treatment. Here we have designed a series of compounds, methyl 5-sulfamoyl-benzoates, which bear a primary sulfonamide group, a well-known marker of CA inhibitors, and determined their affinities for all twelve CA isozymes. Variations of substituents on the benzenesulfonamide ring led to compound 4b , which exhibited an extremely high observed binding affinity to CAIX; the K d was 0.12 nM. The intrinsic dissociation constant, where the binding-linked protonation reactions have been subtracted, reached 0.08 pM. The compound also exhibited more than 100-fold selectivity over the remaining CA isozymes. The X-ray crystallographic structure of compound 3b bound to CAIX showed the structural position, while several structures of compounds bound to other CA isozymes showed structural reasons for compound selectivity towards CAIX. Since this series of compounds possess physicochemical properties suitable for drugs, they may be developed for anticancer therapeutic purposes.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses mRNA capping to evade the human immune system. The cap formation is performed by the SARS-CoV-2 mRNA cap methyltransferases (MTases) nsp14 and nsp16, which are emerging targets for the development of broad-spectrum antiviral agents. Here, we report results from high-throughput virtual screening against these two enzymes. The docking of seven million commercially available drug-like compounds and S-adenosylmethionine (SAM) co-substrate analogues against both MTases resulted in 80 virtual screening hits (39 against nsp14 and 41 against nsp16), which were purchased and tested using an enzymatic homogeneous time-resolved fluorescent energy transfer (HTRF) assay. Nine compounds showed micromolar inhibition activity (IC 50 < 200 μM). The selectivity of the identified inhibitors was evaluated by cross-checking their activity against human glycine N-methyltransferase. The majority of the compounds showed poor selectivity for a specific MTase, no cytotoxic effects, and rather poor cell permeability. Nevertheless, the identified compounds represent good starting points that have the potential to be developed into efficient viral MTase inhibitors.

MERS-CoV continues to cause human outbreaks, so far in 27 countries worldwide following the first registered epidemic in Saudi Arabia in 2012. In this study, we produced a nanovaccine based on virus-like particles (VLPs). VLPs are safe vaccine platforms as they lack any replication-competent genetic material, and are used since many years against hepatitis B virus (HBV), hepatitis E virus (HEV) and human papilloma virus (HPV). In order to produce a vaccine that is readily scalable, we genetically fused the receptor-binding motif (RBM) of MERS-CoV spike protein into the surface of cucumber-mosaic virus VLPs. The employed CuMV TT -VLPs represent a new immunologically optimized vaccine platform incorporating a universal T cell epitope derived from tetanus toxin (TT). The resultant vaccine candidate (mCuMV TT -MERS) is a mosaic particle and consists of unmodified wild type monomers and genetically modified monomers displaying RBM, co-assembling within E. coli upon expression. mCuMV TT -MERS vaccine is self-adjuvanted with ssRNA, a TLR7/8 ligand which is spontaneously packaged during the bacterial expression process. The developed vaccine candidate induced high anti-RBD and anti-spike antibodies in a murine model, showing high binding avidity and an ability to completely neutralize MERS-CoV/EMC/2012 isolate, demonstrating the protective potential of the vaccine candidate for dromedaries and humans., (© 2021. The Author(s).)

Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymatic processes, such as carbon fixation, choline, ethanolamine and propanediol degradation, and others. Formed of a semi-permeable protein shell and an enzymatic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to encapsulate non-native enzymes, BMCs show high potential for applied use. For this goal, a detailed look into shell form variability is significant to predict shell adaptability. Here we present four novel 3D cryo-EM maps of recombinant Klebsiella pneumoniae GRM2 BMC shell particles with the resolution in range of 9 to 22 Å and nine novel 2D classes corresponding to discrete BMC shell forms. These structures reveal icosahedral, elongated, oblate, multi-layered and polyhedral traits of BMCs, indicating considerable variation in size and form as well as adaptability during shell formation processes., (© 2021 The Protein Society.)

COVID-19 is a novel disease caused by SARS-CoV-2 which has conquered the world rapidly resulting in a pandemic that massively impacts our health, social activities, and economy. It is likely that vaccination is the only way to form "herd immunity" and restore the world to normal. Here we developed a vaccine candidate for COVID-19 based on the virus-like particle AP205 displaying the spike receptor binding motif (RBM), which is the major target of neutralizing antibodies in convalescent patients. To this end, we genetically fused the RBM domain of SARS-CoV-2 to the C terminus of AP205 of dimerized capsid proteins. The fused VLPs were expressed in E. coli , which resulted in insoluble aggregates. These aggregates were denatured in 8 M urea followed by refolding, which reconstituted VLP formation as confirmed by electron microscopy analysis. Importantly, immunized mice were able to generate high levels of IgG antibodies recognizing eukaryotically expressed receptor binding domain (RBD) as well as spike protein of SARS-CoV-2. Furthermore, induced antibodies were able to neutralize SARS-CoV-2/ABS/NL20. Additionally, this vaccine candidate has the potential to be produced at large scale for immunization programs.

In the design of high-affinity and enzyme isoform-selective inhibitors, we applied an approach of augmenting the substituents attached to the benzenesulfonamide scaffold in three ways, namely, substitutions at the 3,5- or 2,4,6-positions or expansion of the condensed ring system. The increased size of the substituents determined the spatial limitations of the active sites of the 12 catalytically active human carbonic anhydrase (CA) isoforms until no binding was observed because of the inability of the compounds to fit in the active site. This approach led to the discovery of high-affinity and high-selectivity compounds for the anticancer target CA IX and antiobesity target CA VB. The x-ray crystallographic structures of compounds bound to CA IX showed the positions of the bound compounds, whereas computational modeling confirmed that steric clashes prevent the binding of these compounds to other isoforms and thus avoid undesired side effects. Such an approach, based on the Lock-and-Key principle, could be used for the development of enzyme-specific drug candidate compounds., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Influenza, an acute, highly contagious respiratory disease, remains a significant threat to public health. More effective vaccination strategies aimed at inducing broad cross-protection not only against seasonal influenza variants, but also zoonotic and emerging pandemic influenza strains are urgently needed. A number of conserved protein targets to elicit such cross-protective immunity have been under investigation, with long alpha-helix (LAH) from hemagglutinin stalk and ectodomain of matrix protein 2 ion channel (M2e) being the most studied ones. Recently, we have reported the three-dimensional structure and some practical applications of LAH expressed in Escherichia coli system (referred to as tri-stalk protein). In the present study, we investigated the immunogenicity and efficacy of a panel of broadly protective influenza vaccine prototypes based on both influenza tri-stalk and triple M2e (3M2e) antigens integrated into phage AP205 virus-like particles (VLPs). While VLPs containing the 3M2e alone induced protection against standard homologous and heterologous virus challenge in mice, only the combination of both conserved influenza antigens into a single VLP fully protected mice from a high-dose homologous H1N1 influenza infection. We propose that a combination of genetic fusion and chemical coupling techniques to expose two different foreign influenza antigens on a single particle is a perspective approach for generation of a broadly-effective vaccine candidate that could protect against the constantly emerging influenza virus strains.

The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine., (Copyright © 2020 American Society for Microbiology.)

A series of new 3-phenyl-5-aryl- N -(4-sulfamoylphenyl)-4,5-dihydro-1 H -pyrazole-1-carboxamide derivatives was designed here, synthesized, and studied for carbonic anhydrase (CAs, EC 4.2.1.1) inhibitory activity against the human (h) isozymes I, II, and VII (cytosolic, off-target isoforms), and IX and XII (anticancer drug targets). Generally, CA I was not effectively inhibited, whereas effective inhibitors were identified against both CAs II (K I s in the range of 5.2-233 nM) and VII (K I s in the range of 2.3-350 nM). Nonetheless, CAs IX and XII were the most susceptible isoforms to this class of inhibitors. In particular, compounds bearing an unsubstituted phenyl ring at the pyrazoline 3 position showed 1.3-1.5 nM K I s against CA IX. In contrast, a subset of derivatives having a 4-halo-phenyl at the same position of the aromatic scaffold even reached subnanomolar K I s against CA XII (0.62-0.99 nM). Docking studies with CA IX and XII were used to shed light on the derivative binding mode driving the preferential inhibition of the tumor-associated CAs. The identified potent and selective CA IX/XII inhibitors are of interest as leads for the development of new anticancer strategies.

Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.

The periplasmic lipoprotein BB0365 of the Lyme disease agent Borrelia burgdorferi is expressed throughout mammalian infection and is essential for all phases of Lyme disease infection; its function, however, remains unknown. In the current study, our structural analysis of BB0365 revealed the same structural fold as that found in the NqrC and RnfG subunits of the NADH:quinone and ferredoxin:NAD + sodium-translocating oxidoreductase complexes, which points to a potential role for BB0365 as a component of the sodium pump. Additionally, BB0365 coordinated Zn 2+ by the His51, His55, His140 residues, and the Zn 2+ -binding site indicates that BB0365 could act as a potential metalloenzyme; therefore, this structure narrows down the potential functions of BB0365, an essential protein for B. burgdorferi to cause Lyme disease., (© 2019 Federation of European Biochemical Societies.)

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO 2 hydration in all living organisms and are actively involved in the regulation of a plethora of pathological and physiological conditions. A set of new coumarin/ dihydrocoumarin derivatives was here synthesized, characterized, and tested as human CA inhibitors. Their inhibitory activity was evaluated against the cytosolic human isoforms hCA I and II and the transmembrane hCA IX and hCA XII. Two compounds showed potent inhibitory activity against hCA IX, being more active or equipotent with the reference drug acetazolamide. Computational procedures were used to investigate the binding mode of this class of compounds within the active site of hCA IX and XII that are validated as anti-tumor targets., Competing Interests: The authors declare no conflict of interest.

Twenty-four novel sulfonamide derivatives incorporating dipeptide tails were synthesized by facile acylation reactions of homosulfanilamide through benzotriazole or dicyclohexyl carbodiimide (DCC) mediated coupling reactions. The carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity of the new compounds was assessed against four human (h) isoforms, hCA I, hCA II, hCA IX and hCA XII. Most of the synthesized compounds showed good in vitro carbonic anhydrase inhibitory properties, with inhibition constants in the low nanomolar range. Particularly, the new dipeptide-sulfonamide conjugates incorporating Ala, Phe and Met in the dipeptide sequence, showed the most effective inhibitory activity against to CA IX and XII., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Long alpha helix (LAH) from influenza virus hemagglutinin (HA) stem or stalk domain is one of the most conserved influenza virus antigens. Expression of N-terminally extended LAH in E. coli leads to assembly of α-h elical homotrimer which is structurally nearly identical to the corresponding region of post-fusion form of native HA. This novel tri-stalk protein was able to differentiate between group 1 and 2 influenza in ELISA with virus-infected mice sera. It was also successfully applied for enzyme-linked immunospot assay to estimate the number of HA stem-reactive antibody (Ab)-secreting cells in mice. An in-house indirect ELISA was developed using a HA tri-stalk protein as a coating antigen for evaluation of HA stem-specific Ab levels in human sera collected in Luxembourg from 211 persons with occupational exposure to swine before the pandemic H1N1/09 virus had spread to Western Europe. Our results show that 70% of these pre-pandemic sera are positive for HA stem-specific Abs. In addition, levels of HA stem-specific Abs have positive correlation with the corresponding IgG titers and neutralizing activities against pandemic H1N1/09 virus., Competing Interests: The authors have declared that no competing interests exist.

Borrelia (B.) bavariensis, B. burgdorferi, B. afzelii, B. garinii, B. spielmanii, and B. mayonii are the causative agents in Lyme disease. Lyme disease spirochetes reside in infected Ixodes ticks and are transferred to mammalian hosts during tick feeding. Once transmitted, spirochetes must overcome the first line of defense of the innate immune system either by binding complement regulators or by terminating the formation of the membrane attack complex (MAC). In B. bavariensis, the proteins BGA66 and BGA71 inhibit complement activation by interacting with the late complement components C7, C8, and C9, as well as with the formed MAC. In this study, we have determined the crystal structure of the potent MAC inhibitor BGA71 at 2.9 Ǻ resolution. The structure revealed a cysteine cross-linked homodimer. Based on the crystal structure of BGA71 and the structure-based sequence alignment with CspA from B. burgdorferi, we have proposed a potential binding site for C7 and C9, both of which are constituents of the formed MAC. Our results shed light on the molecular mechanism of immune evasion developed by the human pathogenic Borrelia species to overcome innate immunity. These results will aid in the understanding of Lyme disease pathogenesis and pave the way for the development of new strategies to prevent Lyme disease.

In this study, new 4-[3-(aryl)-5-substitutedphenyl-4,5-dihydro-1H-pyrazole-1-yl]benzensulfonamides (19-36) were synthesized and evaluated their cytotoxic/anticancer and CA inhibitory effects. According to results obtained, the compounds 34 (4-[5-(2,3,4-trimethoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-yl] benzensulfonamide, Potency-Selectivity Expression (PSE) = 141) and 36 (4-[5-(3,4,5-trimethoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-yl]benzensulfonamide, PSE = 54.5) were found the leader anticancer compounds with the highest PSE values. In CA inhibitory studies, the compounds 36 and 24 (4-[5-(3,4,5-trimethoxyphenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-1-yl]benzensulfonamide) were found the leader CA inhibitors depending on selectivity ratios. The compound 36 was a selective inhibitor of hCA XII isoenzyme (hCA I/hCA XII = 1250 and hCA II/hCA XII = 224) while the compound 24 was a selective inhibitor of hCA IX isoenzyme (hCA I/hCA IX = 161 and hCA II/hCA IX = 177). The compounds 24, 34, and 36 can be considered to develop new anticancer drug candidates., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the most common tick-borne disease in the US and Europe. No potent human vaccine is currently available. The innate immune complement system is vital to host defense against pathogens, as complement activation on the surface of spirochetes results in bacterial killing. Complement system is inhibited by the complement regulator factor H (FH). To escape killing, B. burgdorferi produces an outer surface protein CspZ that binds FH to inhibit complement activation on the cell surface. Immunization with CspZ alone does not protect mice from infection, which we speculate is because FH-binding cloaks potentially protective epitopes. We modified CspZ by conjugating to virus-like particles (VLP-CspZ) and eliminating FH binding (modified VLP-CspZ) to increase immunogenicity. We observed greater bactericidal antibody titers in mice vaccinated with modified VLP-CspZ: A serum dilution of 1:395 (modified VLP-CspZ) vs 1:143 (VLP-CspZ) yielded 50% borreliacidal activity. Immunizing mice with modified VLP-CspZ cleared spirochete infection, as did passive transfer of elicited antibodies. This work developed a novel Lyme disease vaccine candidate by conjugating CspZ to VLP and eliminating FH-binding ability. Such a strategy of conjugating an antigen to a VLP and eliminating binding to the target ligand can serve as a general model for developing vaccines against other bacterial infectious agents.

A new chemotype with carbonic anhydrase (CA, EC 4.2.1.1) inhibitory action has been discovered, the homo-sulfocoumarins (3H-1,2-benzoxathiepine 2,2-dioxides) which have been designed considering the (sulfo)coumarins as lead molecules. An original synthetic strategy of a panel of such derivatives led to compounds with a unique inhibitory profile and very high selectivity for the inhibition of the tumour associated (CA IX/XII) over the cytosolic (CA I/II) isoforms. Although the CA inhibition mechanism with these new compounds is unknown for the moment, we hypothesize that it may be similar to that of the sulfocoumarins, i.e. hydrolysis to the corresponding sulfonic acids which thereafter anchor to the zinc-coordinated water molecule within the enzyme active site.

Modern DNA sequencing capabilities have led to the discovery of a large number of new bacteriophage genomes, which are a rich source of novel proteins with an unidentified biological role. The genome of Enterobacter cancerogenus bacteriophage Enc34 contains several proteins of unknown function that are nevertheless conserved among distantly related phages. Here, we report the crystal structure of a conserved Enc34 replication protein ORF6 which contains a domain of unknown function DUF2815. Despite the low (~15%) sequence identity, the Enc34 ORF6 structurally resembles the gene 2.5 protein from bacteriophage T7, and likewise is a single-stranded DNA (ssDNA)-binding protein (SSB) that consists of a variation of the oligosaccharide/oligonucleotide-binding (OB)-fold and an unstructured C-terminal segment. We further report the crystal structure of a C-terminally truncated ORF6 in complex with an ssDNA oligonucleotide that reveals a DNA-binding mode involving two aromatic stacks and multiple electrostatic interactions, with implications for a common ssDNA recognition mechanism for all T7-type SSBs.

Background: The lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutinin (HA) stalk regions. For an improved immune response, the LAH domain from H3N2 strain has been incorporated into virus-like particles (VLPs) derived from hepatitis B virus core protein (HBc) using recently developed tandem core technology., Results: Fermentation conditions for recombinant HBc-LAH were established in yeast Pichia pastoris and a rapid and efficient purification method for chimeric VLPs was developed to match the requirements for industrial scale-up. Purified VLPs induced strong antibody responses against both group 1 and group 2 HA proteins in mice., Conclusion: Our results indicate that the tandem core technology is a useful tool for incorporation of highly hydrophobic LAH domain into HBc VLPs. Chimeric VLPs can be successfully produced in bioreactor using yeast expression system. Immunologic data indicate that HBc VLPs carrying the LAH antigen represent a promising universal influenza vaccine component.

A series of N -acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids ( EMAC8000a-m ) was designed and synthesized with the aim to target tumor associated carbonic anhydrase (hCA) isoforms IX and XII. Most of the compounds were selective inhibitors of the tumor associated hCA XII. Moreover, resolution of EMAC8000d racemic mixture led to the isolation of the levorotatory eutomer exhibiting an increase of hCA XII inhibition potency and selectivity with respect to hCA II. Computational studies corroborated these data. Overall our data indicate that both substitution pattern and stereochemistry of dihydro-1,3,4-oxadiazole could be considered as key factors to determine activity and selectivity toward hCA isozymes. These results can provide further indication for the design and optimization of selective hCA inhibitors.

Plasmepsin IV from Plasmodium falciparum (PM IV) is a promising target for the development of novel antimalarial drugs. Here, the crystal structure of the truncated zymogen of PM IV (pPM IV), consisting of the mature enzyme plus a prosegment of 47 residues, has been determined at 1.5 Å resolution. pPM IV presents the fold previously described for studied proplasmepsins, displaying closer similarities to proplasmepin IV from P. vivax (pPvPM) than to the other two proplasmepsins from P. falciparum. The study and comparison of the pPM IV structure with the proplasmepsin structures described previously provide information about the similarities and differences in the inactivation-activation mechanisms among the plasmepsin zymogens.

In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that in situ, the viral RNA genome can adopt a defined conformation. The RNA forms a branched network of stem-loops that almost all allocate near the capsid inner surface, while predominantly binding to coat protein dimers that are located in one-half of the capsid. This suggests that genomic RNA is highly involved in genome packaging and virion assembly.

Protein structure determination by proton-detected magic-angle spinning (MAS) NMR has focused on highly deuterated samples, in which only a small number of protons are introduced and observation of signals from side chains is extremely limited. Here, we show in two fully protonated proteins that, at 100-kHz MAS and above, spectral resolution is high enough to detect resolved correlations from amide and side-chain protons of all residue types, and to reliably measure a dense network of (1)H-(1)H proximities that define a protein structure. The high data quality allowed the correct identification of internuclear distance restraints encoded in 3D spectra with automated data analysis, resulting in accurate, unbiased, and fast structure determination. Additionally, we find that narrower proton resonance lines, longer coherence lifetimes, and improved magnetization transfer offset the reduced sample size at 100-kHz spinning and above. Less than 2 weeks of experiment time and a single 0.5-mg sample was sufficient for the acquisition of all data necessary for backbone and side-chain resonance assignment and unsupervised structure determination. We expect the technique to pave the way for atomic-resolution structure analysis applicable to a wide range of proteins., Competing Interests: The authors declare no conflict of interest.

Plasmepsin II (PMII) is one of the ten plasmepsins (PMs) identified in the genome of Plasmodium falciparum, the causative agent of the most severe and deadliest form of malaria. Owing to the emergence of P. falciparum strains that are resistant to current antimalarial agents such as chloroquine and sulfadoxine/pyrimethamine, there is a constant pressure to find new and lasting chemotherapeutic drug therapies. Previously, the crystal structure of PMII in complex with NU655, a potent antimalarial hydroxyethylamine-based inhibitor, and the design of new compounds based on it have been reported. In the current study, two of these newly designed hydroxyethylamine-based inhibitors, PG418 and PG394, were cocrystallized with PMII and their structures were solved, analyzed and compared with that of the PMII-NU655 complex. Structural analysis of the PMII-PG418 complex revealed that the flap loop can adopt a fully closed conformation, stabilized by interactions with the inhibitor, and a fully open conformation, causing an overall expansion in the active-site cavity, which in turn causes unstable binding of the inhibitor. PG418 also stabilizes the flexible loop Gln275-Met286 of another monomer in the asymmetric unit of PMII, which is disordered in the PMII-NU655 complex structure. The crystal structure of PMII in complex with the inhibitor PG418 demonstrates the conformational flexibility of the active-site cavity of the plasmepsins. The interactions of the different moieties in the P1' position of PG418 and PG394 with Thr217 have to be taken into account in the design of new potent plasmepsin inhibitors.

CutC choline trimethylamine-lyase is an anaerobic bacterial glycyl radical enzyme (GRE) that cleaves choline to produce trimethylamine (TMA) and acetaldehyde. In humans, TMA is produced exclusively by the intestinal microbiota, and its metabolite, trimethylamine oxide, has been associated with a higher risk of cardiovascular diseases. Therefore, information about the three-dimensional structures of TMA-producing enzymes is important for microbiota-targeted drug discovery. We have cloned, expressed, and purified the CutC GRE and the activating enzyme CutD from Klebsiella pneumoniae, a representative of the human microbiota. We have determined the first crystal structures of both the choline-bound and choline-free forms of CutC and have discovered that binding of choline at the ligand-binding site triggers conformational changes in the enzyme structure, a feature that has not been observed for any other characterized GRE., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Borrelia burgdorferi is the causative agent of Lyme disease and is found in two different types of hosts in nature - Ixodes ticks and various mammalian organisms. To initiate disease and survive in mammalian host organisms, B. burgdorferi must be able to transfer to a new host, proliferate, attach to different tissue and resist the immune response. To resist the host's immune response, B. burgdorferi produces at least five different outer surface proteins that can bind complement regulator factor H (CFH) and/or factor H-like protein 1 (CFHL-1). The crystal structures of two uniquely folded complement binding proteins, which belong to two distinct gene families and are not found in other bacteria, have been previously described. The crystal structure of the CFH and CFHL-1 binding protein CspZ (also known as BbCRASP-2 or BBH06) from B. burgdorferi, which belongs to a third gene family, is reported in this study. The structure reveals that the overall fold is different from the known structures of the other complement binding proteins in B. burgdorferi or other bacteria; this structure does not resemble the fold of any known protein deposited in the Protein Data Bank. The N-terminal part of the CspZ protein forms a four-helix bundle and has features similar to the FAT domain (focal adhesion targeting domain) and a related domain found in the vinculin/α-catenin family. By combining our findings from the crystal structure of CspZ with previous mutagenesis studies, we have identified a likely binding surface on CspZ for CFH and CFHL-1., (© 2014 FEBS.)

Antimalarial hit 1 SR (TCMDC-134674) identified in a GlaxoSmithKline cell based screening campaign was evaluated for inhibitory activity against the digestive vacuole plasmepsins (Plm I, II, and IV). It was found to be a potent Plm IV inhibitor with no selectivity over Cathepsin D. A cocrystal structure of 1 SR bound to Plm II was solved, providing structural insight for the design of more potent and selective analogues. Structure-guided optimization led to the identification of structurally simplified analogues 17 and 18 as low nanomolar inhibitors of both, plasmepsin Plm IV activity and P. falciparum growth in erythrocytes.

We present here (1)H-detected triple-resonance H/N/C experiments that incorporate CO-CA and CA-CB out-and-back scalar-transfer blocks optimized for robust resonance assignment in biosolids under ultra-fast magic-angle spinning (MAS). The first experiment, (H)(CO)CA(CO)NH, yields (1)H-detected inter-residue correlations, in which we record the chemical shifts of the CA spins in the first indirect dimension while during the scalar-transfer delays the coherences are present only on the longer-lived CO spins. The second experiment, (H)(CA)CB(CA)NH, correlates the side-chain CB chemical shifts with the NH of the same residue. These high sensitivity experiments are demonstrated on both fully-protonated and 100%-H(N) back-protonated perdeuterated microcrystalline samples of Acinetobacter phage 205 (AP205) capsids at 60 kHz MAS.

Background: Bacteriophages of the Leviviridae family are small RNA viruses with linear, positive-sense, single-stranded RNA genomes that encode only four proteins. All phages of this family require bacterial pili to attach to and infect cells. Leviviridae phages utilizing F-pili for this purpose have been extensively studied. RNA phages specific for conjugative plasmid-encoded pili other than that of plasmid F have been isolated, but are much less understood and their relation to the F-pili-specific phages in many cases is not known., Results: Phage M has the smallest known Leviviridae genome to date and has the typical genome organization with maturation, coat and replicase genes in the 5' to 3' direction. The lysis gene is located in a different position than in other known Leviviridae phages and completely overlaps with the replicase gene in a different reading frame. It encodes a 37 residue long polypeptide that contains a transmembrane helix like the other known lysis proteins of leviviruses. Sequence identities of M proteins to those of other phages do not exceed 25% for maturation protein, 51% for coat protein and 41% for replicase. Similarities in protein sequences and RNA secondary structures at the 3' untranslated region place phage M together with phages specific for IncP, IncC and IncH, but not IncF plasmid-encoded pili. Phylogenetic analysis using the complete genome sequences and replicase proteins suggests that phage M represents a lineage that branched off early in the course of RNA phage specialization on different conjugative plasmids., Conclusions: The genome sequence of phage M shows that it is clearly related to other conjugative pili-specific leviviruses but has an atypical location of the lysis gene. It provides a better view on the remarkable diversification of the plasmid-specific RNA phages.