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The proline-rich antimicrobial peptide (PrAMP) Drosocin (Dro) from fruit flies shows sequence similarity to other PrAMPs that bind to the ribosome and inhibit protein synthesis by varying mechanisms. The target and mechanism of action of Dro, however, remain unknown. Here we show that Dro arrests ribosomes at stop codons, probably sequestering class 1 release factors associated with the ribosome. This mode of action is comparable to that of apidaecin (Api) from honeybees, making Dro the second member of the type II PrAMP class. Nonetheless, analysis of a comprehensive library of endogenously expressed Dro mutants shows that the interactions of Dro and Api with the target are markedly distinct. While only a few C-terminal amino acids of Api are critical for binding, the interaction of Dro with the ribosome relies on multiple amino acid residues distributed throughout the PrAMP. Single-residue substitutions can substantially enhance the on-target activity of Dro.
Assuntos
Peptídeos Antimicrobianos , Biossíntese de Proteínas , Animais , Escherichia coli/metabolismo , Glicopeptídeos/química , Drosophila/química , Drosophila/metabolismoRESUMO
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered the worldwide coronavirus disease 2019 (COVID-19) pandemic. Serological assays for the detection of SARS-CoV-2 infections are important to understand the immune response in patients and to obtain epidemiological data about the number of infected people, especially to identify asymptomatic persons not aware of a past infection. METHODS: We recombinantly produced SARS-CoV-2 nucleocapsid (N)-protein in Escherichia coli. We used the purified protein to develop an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV-2 specific antibodies. This ELISA method was optimized and validated with serum samples collected from 113 patients with RT-PCR-confirmed SARS-CoV-2 infections including hospitalized COVID-19 patients and 1500 control sera mostly collected before 2015 with different clinical background. RESULTS: The optimized N-protein-ELISA provided a sensitivity of 89.7% (n = 68) for samples collected from patients with confirmed SARS-CoV-2 infections and mild to severe symptoms more than 14 days after symptom onset or a positive PCR test. The antibody levels remained low for serum samples collected in the first six days (n = 23) and increased in the second week (n = 22) post symptom onset or PCR confirmation. At this early phase, the ELISA provided a sensitivity of 39.1% and 86.4%, respectively, reflecting the time of an IgG immune response against pathogens. The assay specificity was 99.3% (n = 1500; 95% CI 0.995-0.999). Serum samples from persons with confirmed antibody titers against human immunodeficiency viruses 1/2, parvovirus B19, hepatitis A/B virus, cytomegalovirus, Epstein Barr virus, and herpes simplex virus were tested negative. CONCLUSIONS: We conclude that the N-protein-based ELISA developed here is well suited for the sensitive and specific serological detection of SARS-CoV-2 specific IgG antibodies in human serum for symptomatic infections. It may also prove useful to identify previous SARS-CoV-2 infections in vaccinated people, as all currently approved vaccines rely on the SARS-CoV-2 spike (S-) protein.
Assuntos
COVID-19 , Infecções por Vírus Epstein-Barr , COVID-19/diagnóstico , Ensaio de Imunoadsorção Enzimática , Herpesvirus Humano 4 , Humanos , Proteínas do Nucleocapsídeo , SARS-CoV-2RESUMO
Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (Kd) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.
Assuntos
Anti-Infecciosos , Escherichia coli , Antibacterianos/química , Anti-Infecciosos/metabolismo , Peptídeos Antimicrobianos , Sítios de Ligação , Escherichia coli/metabolismo , Prolina/metabolismo , Ribossomos/metabolismoRESUMO
This study proposes an innovative strategy to enhance the pharmacophore model of antimicrobial bismuth thiolato complex drugs by substituting hydrocarbon ligand structures with boron clusters, particularly icosahedral closo-dicarbadodecaborane (C2B10H12, carboranes). The hetero- and homoleptic mercaptocarborane complexes BiPh2L (1) and BiL3 (2) (L=9-S-1,2-C2B10H11) were prepared from 9-mercaptocarborane (HL) and triphenylbismuth. Comprehensive characterization using NMR, IR, MS, and XRD techniques confirmed their successful synthesis. Evaluation of antimicrobial activity in a liquid broth microdilution assay demonstrated micromolar to submicromolar minimum inhibitory concentrations (MIC) suggesting high effectiveness against S.â aureus and limited efficacy against E.â coli. This study highlights the potential of boron-containing bismuth complexes as promising antimicrobial agents, especially targeting Gram-positive bacteria, thus contributing to the advancement of novel therapeutic approaches.
Assuntos
Antibacterianos , Bismuto , Escherichia coli , Testes de Sensibilidade Microbiana , Staphylococcus aureus , Bismuto/química , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Desenho de Fármacos , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/síntese química , Boranos/química , Boranos/farmacologia , Compostos de Sulfidrila/química , Estrutura Molecular , Relação Estrutura-Atividade , Compostos de Boro/química , Compostos de Boro/farmacologia , HumanosRESUMO
Background: Cryptococcosis and cryptococcal meningitis, caused by Cryptococcus neoformans infections, lead to approximately 180,000 deaths per year, primarily in developing countries. Individuals with compromised immune systems, e.g., due to HIV infection (AIDS) or chemotherapy, are particularly vulnerable. Conventional treatment options are often limited and can cause severe side effects. Therefore, this study aimed to investigate the antifungal effect of insect-derived proline-rich antimicrobial peptides (PrAMPs) against C. neoformans. These peptides are known for their low toxicity and their high efficacy in murine infection models, making them a promising alternative for treatment. Results: A preliminary screening of the minimal inhibitory concentrations (MICs) of 20 AMPs, including the well-known PrAMPs Onc112, Api137, and Chex1Arg20 as well as the cathelicidin CRAMP against the C. neoformans strains 1841, H99, and KN99α revealed promising results, with MICs as low as 1.6 µmol/L. Subsequent investigations of selected peptides, determining their influence on fungal colony-forming units, confirmed their strong activity. The antifungal activity was affected by factors such as peptide net charge and sequence, with stronger effects at higher net charges probably due to better intracellular uptake confirmed by confocal laser scanning microscopy. Inactive scrambled peptides suggest a specific intracellular target, although scanning electron microscopy showed that PrAMPs also damaged the cell exterior for a low proportion of the cells. Possible pore formation could facilitate entry into the cytosol.
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The antimicrobial peptide (AMP) ARV-1502 was designed based on naturally occurring short proline-rich AMPs, including pyrrhocoricin and drosocin. Identification of chaperone DnaK as a therapeutic target in Escherichia coli triggered intense research on the ligand-DnaK-interactions using fluorescence polarization and X-ray crystallography to reveal the binding motif and characterize the influence of the chaperone on protein refolding activity, especially in stress situations. In continuation of this research, 182 analogs of ARV-1502 were designed by substituting residues involved in antimicrobial activity against Gram-negative pathogens. The peptides synthesized on solid-phase were examined for their binding to E. coli and S. aureus DnaK providing 15 analogs with improved binding characteristics for at least one DnaK. These 15 analogs were distinguished from the original sequence by their increased hydrophobicity parameters. Additionally, the influence of the entire DnaK chaperone system, including co-chaperones DnaJ and GrpE on refolding and ATPase activity, was investigated. The increasingly hydrophobic peptides showed a stronger inhibitory effect on the refolding activity of E. coli chaperones, reducing protein refolding by up to 64%. However, these more hydrophobic peptides had only a minor effect on the ATPase activity. The most dramatic changes on the ATPase activity involved peptides with aspartate substitutions. Interestingly, these peptides resulted in a 59% reduction of the ATPase activity in the E. coli chaperone system whereas they stimulated the ATPase activity in the S. aureus system up to 220%. Of particular note is the improvement of the antimicrobial activity against S. aureus from originally >128 µg/mL to as low as 16 µg/mL. Only a single analog exhibited improved activity over the original value of 8 µg/mL against E. coli. Overall, the various moderate-throughput screenings established here allowed identifying (un)favored substitutions on 1) DnaK binding, 2) the ATPase activity of DnaK, 3) the refolding activity of DnaK alone or together with co-chaperones, and 4) the antimicrobial activity against both E. coli and S. aureus.
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The rapid development, approval, and production of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than 1 year after the first reports of a new infectious disease was a real game changer, providing 80%-90% efficacy in preventing severe etiopathologies of the coronavirus disease 2019 (COVID-19). These vaccines induce an immune response against the SARS-CoV-2 spike (S) protein located on the surface of the virus particle. Antibodies (Abs) recognizing the S-protein can inhibit binding of the virus via the S-protein to the angiotensin-converting enzyme-2 (ACE-2) receptor expressed on different human cells, especially when these Abs bind to the interaction site, the so-called receptor-binding domain (RBD). We have expressed the RBDs of wild-type SARS-CoV-2 and five variants of concern (VOCs) to test the immune response in people before vaccination with mRNA vaccines BNT162b2 and mRNA-1273 and after up to three vaccinations using in-house ELISA and inhibition assays. The methods of both assays are provided. Both vaccines initiated similarly high IgG titers after two vaccinations against the wild-type and even two VOC-RBDs (alpha and delta) and strongly inhibited the corresponding RBD-ACE-2 binding. The IgG titers and inhibition of ACE-2 binding were lower for beta and gamma RBDs and much lower for omicron RBD. The third vaccination after 6 months strongly increased both the IgG titers and the neutralizing effect against all variants, especially for omicron, leading to 63% ± 13% neutralization potential. Importantly, neutralization linearly increased with the IgG titers.
Assuntos
COVID-19 , SARS-CoV-2 , Vacina BNT162 , COVID-19/prevenção & controle , Humanos , Imunoglobulina G , RNA MensageiroRESUMO
This study investigated the IgG and IgA antibody response against recombinant S1 and receptor binding domains (RBD) of the spike (S-) protein and the membrane (M-) protein using a set of 115 serum samples collected from patients infected with SARS-CoV-2 in Germany before April 2021 using protein and peptide ELISA. As S1- and RBD-proteins expressed in Escherichia coli provided poor sensitivities in ELISA, they were replaced by proteins expressed in HEK cells. The RBD-ELISA provided a sensitivity of 90.6% (N = 85) for samples collected from patients with confirmed SARS-CoV-2 infections more than 14 days after symptom onset or a positive PCR test. In population-based controls, the specificity was 97.9% (N = 94). In contrast, the sensitivities were only 41.2% and 72.6% for M- and N-proteins, respectively, while the specificities were 88.5% and 100%, respectively. Considering also 20 samples collected during the first two weeks of symptom onset or PCR confirmation, the sensitivity of RBD- and N-protein ELISA decreased to 82.6% and 72.6%, respectively. The combination of two data sets, i.e., N- and RBD-, N- and M-, or RBD- and M-proteins increased the sensitivity to 85.8%, 77.9%, and 87.8%, respectively. Peptide mapping mostly confirmed epitopes previously reported for S1- and M-proteins, but they were only recognized by a few samples already tested positive in the corresponding protein ELISA indicating that peptide-based assays will not improve the diagnostic sensitivity.
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The designer proline-rich antimicrobial peptide (PrAMP) Chex1-Arg20 amide (ARV-1502) is active against Gram-negative and Gram-positive pathogens in different murine infection models when administered parenterally and possesses a wide therapeutic index. Here we studied the pharmacokinetics of ARV-1502 for the first time when administered intramuscularly or intravenously (IV) in Sprague Dawley rats and Beagle dogs. First, a specific and robust quantitation method relying on parallel reaction monitoring (PRM) using a high-resolution hybrid quadrupole-Orbitrap mass spectrometer coupled on-line to reversed-phase uHPLC was established and validated. The limit of detection was 2 ng/mL and the limit of quantitation was 4 ng/mL when spiked to pooled rat and dog plasma. When ARV-1502 was administered IV at doses of 75 and 250 µg/kg in dogs and rats, the plasma concentrations were 0.7 and 3.4 µg/mL 2 min post-administration, respectively. ARV-1502 plasma concentrations declined exponentially reaching levels between 2 and 4 ng/mL after 2 h. Intramuscular administration of 0.75 mg/kg in dogs and 2.5 mg/kg in rats resulted in a different pharmacokinetics profile. The plasma concentrations peaked at 15 min post-injection at 1 µg/mL (dogs) and 12 µg/mL (rats) and decreased exponentially within 3 h to 4 and 16 ng/mL, respectively. The initial plasma concentrations of ARV-1502 and the decay timing afterwards indicated that the peptide circulated in the blood stream for several hours, at some point above the minimal inhibitory concentration against multidrug-resistant Enterobacteriaceae, with blood concentrations sufficient to suppress bacterial growth and to modulate the immune system.