RESUMO
Apd1, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxin-like ferredoxin (TLF) domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi, and unicellular pathogenic eukaryotes but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apd1 and Aim32 proteins are essential for viability of yeast cells upon treatment with the redox mediators gallobenzophenone or pyrogallol, respectively. UV-vis, EPR, and Mössbauer spectroscopy of purified wild-type Apd1 and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion, and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S]2+/1+ cluster. The [2Fe-2S] center of Apd1 ( Em,7 = -164 ± 5 mV, p Kox1,2 = 7.9 ± 0.1 and 9.7 ± 0.1) differs from both dioxygenase ( Em,7 ≈ -150 mV, p Kox1,2 = 9.8 and 11.5) and cytochrome bc1/ b6 f Rieske clusters ( Em,7 ≈ +300 mV, p Kox1,2= 7.7 and 9.8). Apd1 and its engineered variants represent an unprecedented flexible system for which a stable [2Fe-2S] cluster with two histidine ligands, (two different) single histidine ligands, or only cysteinyl ligands is possible in the same protein fold. Our results define a remarkable example of convergent evolution of the [2Fe-2S] cluster containing proteins with bishistidinyl coordination.
Assuntos
Ferredoxinas/química , Ferredoxinas/metabolismo , Histidina , Transporte de Elétrons , Domínios ProteicosRESUMO
PURPOSE: Direct visualization is a very effective method in accomplishing adequate articular surface reconstruction in fracture repair. This study investigates distal tibial plafond articular surface visibility using the anteromedial, anterolateral, posteromedial, and posterolateral approaches, the effect of instrumented distraction on visibility, and which zones of the articular surface are visible for each approach. METHODS: The anteromedial, anterolateral, posteromedial, and posterolateral approaches to the distal tibial plafond were performed on 16 cadaveric ankle specimens. The articular surface visualization for each approach was marked using an electrocautery device with manual and instrumented distraction. Articular surface visualization was photographically documented. Digital axial segmentation and quantitative analysis of the visualized distal tibial plafond articular surface were performed. RESULTS: With manual distraction, distal tibial plafond articular surface visualization, expressed in percent of overall articular surface, was limited to 9% (SD ± 9) for the anteromedial, 24% (SD ± 18) for the anterolateral, 26% (SD ± 10) for the posteromedial, and 30% (SD ± 18) for the posterolateral approaches. Using instrumented distraction significantly improved articular surface visualization in all instances (p < 0.001). The anteromedial approach visible articular surface increased to 63% (SD ± 13), the anterolateral to 72% (SD ± 22), the posteromedial to 62% (SD ± 11), and the posterolateral to 50% (± 17). CONCLUSION: This study demonstrates the efficacy of instrumented distraction when attempting surgical visualization of the distal tibial plafond articular surface. Knowledge of approach specific articular surface visibility may assist the surgeon in choosing the appropriate approach(es) based on case-specific distal tibial plafond fracture patterns. LEVEL OF EVIDENCE: IV, cadaver study.
Assuntos
Fraturas do Tornozelo , Fraturas da Tíbia , Fraturas do Tornozelo/cirurgia , Articulação do Tornozelo/diagnóstico por imagem , Articulação do Tornozelo/cirurgia , Cadáver , Fixação Interna de Fraturas/métodos , Humanos , Tíbia/diagnóstico por imagem , Tíbia/cirurgia , Fraturas da Tíbia/diagnóstico por imagem , Fraturas da Tíbia/cirurgiaRESUMO
Small macrocyclic peptides are promising candidates for new anti-infective drugs. To date, such peptides have been poorly studied in the context of anti-virulence targets. Using phage display and a self-designed peptide library, we identified a cyclic heptapeptide that can bind the carbon storage regulator A (CsrA) from Yersinia pseudotuberculosis and displace bound RNA. This disulfide-bridged peptide, showed an IC50 value in the low micromolar range. Upon further characterization, cyclisation was found to be essential for its activity. To increase metabolic stability, a series of disulfide mimetics were designed and a redox-stable 1,4-disubstituted 1,2,3-triazole analogue displayed activity in the double-digit micromolar range. Further experiments revealed that this triazole peptidomimetic is also active against CsrA from Escherichia coli and RsmA from Pseudomonas aeruginosa. This study provides an ideal starting point for medicinal chemistry optimization of this macrocyclic peptide and might pave the way towards broad-acting virulence modulators.
Assuntos
Bacteriófagos , Peptídeos Cíclicos , Carbono , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Pseudomonas aeruginosa/metabolismo , VirulênciaRESUMO
Phage display is a commonly used technology for the screening of large clonal libraries of proteins and peptides. The construction of peptide libraries containing very short sequences, however, poses certain problems for conventional restriction-based cloning procedures, which are rooted in the necessity to purify restricted library oligos. Herein, we present an alternative cloning method especially suitable for such very short sequences of about only 21 base pairs resulting in a 60 bp insert. The employed restriction-free hot fusion cloning strategy allows for facile library construction bypassing the need for purification of the small oligo. The library includes one well-defined disulfide bridge rendering the displayed macrocyclic peptide sequences as attractive scaffolds for novel active principles.
Assuntos
Escherichia coli , Compostos Macrocíclicos , Biblioteca de Peptídeos , Engenharia de Proteínas , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
With the aim to develop novel antiviral agents against Kaposi's Sarcoma Herpesvirus (KSHV), we are targeting the latency-associated nuclear antigen (LANA). This protein plays an important role in viral genome maintenance during latent infection. LANA has the ability to tether the viral genome to the host nucleosomes and, thus, ensures latent persistence of the viral genome in the host cells. By inhibition of the LANA-DNA interaction, we seek to eliminate or reduce the load of the viral DNA in the host. To achieve this goal, we screened our in-house library using a dedicated fluorescence polarization (FP)-based competition assay, which allows for the quantification of LANA-DNA-interaction inhibition by small organic molecules. We successfully identified three different compound classes capable of disrupting this protein-nucleic acid interaction. We characterized these compounds by IC50 dose-response evaluation and confirmed the compound-LANA interaction using surface plasmon resonance (SPR) spectroscopy. Furthermore, two of the three hit scaffolds showed only marginal cytotoxicity in two human cell lines. Finally, we conducted STD-NMR competition experiments with our new hit compounds and a previously described fragment-sized inhibitor. Based on these results, future compound linking approaches could serve as a promising strategy for further optimization studies in order to generate highly potent KSHV inhibitors.
Assuntos
Antivirais/farmacologia , Herpesvirus Humano 8/efeitos dos fármacos , Proteínas Nucleares/antagonistas & inibidores , Antígenos Virais/metabolismo , Antivirais/toxicidade , DNA/metabolismo , Descoberta de Drogas , Células HEK293 , Células Hep G2 , Humanos , Testes de Sensibilidade Microbiana , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/toxicidadeRESUMO
The Latency-associated nuclear antigen (LANA) plays a central role for the latent persistence of the Kaposi's Sarcoma Herpesvirus (KSHV) in the human host and helps to establish lifelong infections. Herein, we report our efforts towards hit-to-lead generation starting from a previously discovered LANA-DNA inhibitor. By tethering the viral genome to the host nucleosomes, LANA ensures the segregation and persistence of the viral DNA during mitosis. LANA is also required for the replication of the latent viral episome during the S phase of the cell cycle. We aim to inhibit the interaction between LANA and the viral genome to prevent the latent persistence of KSHV in the host organism. Medicinal chemistry-driven optimization studies and structure-activity-relationship investigation led to the discovery of an improved LANA inhibitor. The functional activity of our compounds was evaluated using a fluorescence polarization (FP)-based interaction inhibition assay and electrophoretic mobility shift assay (EMSA). Even though a crystal structure of the ligand protein complex was not available, we successfully conducted hit optimization toward a low micromolar protein-nucleic acid-interaction inhibitor. Additionally, we applied STD-NMR studies to corroborate target binding and to gain insights into the binding orientation of our most potent inhibitor, providing opportunities for further rational design of more efficient LANA-targeting anti KSHV agents in future studies.
Assuntos
Antivirais/farmacologia , Infecções por Herpesviridae/tratamento farmacológico , Herpesvirus Humano 8/efeitos dos fármacos , Isoquinolinas/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Triazóis/farmacologia , Antígenos Virais/metabolismo , Antivirais/síntese química , Antivirais/química , DNA Viral/efeitos dos fármacos , Relação Dose-Resposta a Droga , Infecções por Herpesviridae/metabolismo , Isoquinolinas/síntese química , Isoquinolinas/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas Nucleares/metabolismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
The latency-associated nuclear antigen (LANA) is required for latent replication and persistence of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8. It acts via replicating and tethering the virus episome to the host chromatin and exerts other functions. We conceived a new approach for the discovery of antiviral drugs to inhibit the interaction between LANA and the viral genome. We applied a biophysical screening cascade and identified the first LANA binders from small, structurally diverse compound libraries. Starting from a fragment-sized scaffold, we generated optimized hits via fragment growing using a dedicated fluorescence-polarization-based assay as the structure-activity-relationship driver. We improved compound potency to the double-digit micromolar range. Importantly, we qualified the resulting hit through orthogonal methods employing EMSA, STD-NMR, and MST methodologies. This optimized hit provides an ideal starting point for subsequent hit-to-lead campaigns providing evident target-binding, suitable ligand efficiencies, and favorable physicochemical properties.