RESUMO
Various vaccines have been challenged by SARS-CoV-2 variants. Here, we reported a yeast-derived recombinant bivalent vaccine (Bivalent wild-type [Wt]+De) based on the wt and Delta receptor-binding domain (RBD). Yeast derived RBD proteins based on the wt and Delta mutant were used as the prime vaccine. It was found that, in the presence of aluminium hydroxide (Alum) and unmethylated CpG-oligodeoxynucleotides (CpG) adjuvants, more cross-protective immunity against SARS-CoV-2 prototype and variants were elicited by bivalent vaccine than monovalent wtRBD or Delta RBD. Furthermore, a heterologous boosting strategy consisting of two doses of bivalent vaccines followed by one dose adenovirus vectored vaccine exhibited cross-neutralization capacity and specific T cell responses against Delta and Omicron (BA.1 and BA.4/5) variants in mice, superior to a homologous vaccination strategy. This study suggested that heterologous prime-boost vaccination with yeast-derived bivalent protein vaccine could be a potential approach to address the challenge of emerging variants.
Assuntos
COVID-19 , Vacinas , Animais , Camundongos , Vacinas Combinadas , Proteínas Fúngicas , Saccharomyces cerevisiae/genética , COVID-19/prevenção & controle , SARS-CoV-2 , VacinaçãoRESUMO
Neurofibromatosis Type II (NF2) is an autosomal dominant cancer predisposition syndrome in which germline haploinsufficiency at the NF2 gene confers a greatly increased propensity for tumor development arising from tissues of neural crest derived origin. NF2 encodes the tumor suppressor, Merlin, and its biochemical function is incompletely understood. One well-established function of Merlin is as a negative regulator of group A serine/threonine p21-activated kinases (PAKs). In these studies we explore the role of PAK1 and its closely related paralog, PAK2, both pharmacologically and genetically, in Merlin-deficient Schwann cells and in a genetically engineered mouse model (GEMM) that develops spontaneous vestibular and spinal schwannomas. We demonstrate that PAK1 and PAK2 are both hyper activated in Merlin-deficient murine schwannomas. In preclinical trials, a pan Group A PAK inhibitor, FRAX-1036, transiently reduced PAK1 and PAK2 phosphorylation in vitro, but had insignificant efficacy in vivo. NVS-PAK1-1, a PAK1 selective inhibitor, had a greater but still minimal effect on our GEMM phenotype. However, genetic ablation of Pak1 but not Pak2 reduced tumor formation in our NF2 GEMM. Moreover, germline genetic deletion of Pak1 was well tolerated, while conditional deletion of Pak2 in Schwann cells resulted in significant morbidity and mortality. These data support the further development of PAK1-specific small molecule inhibitors and the therapeutic targeting of PAK1 in vestibular schwannomas and argue against PAK1 and PAK2 existing as functionally redundant protein isoforms in Schwann cells.
Assuntos
Neurofibromatose 2/genética , Quinases Ativadas por p21/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Genes Supressores de Tumor/efeitos dos fármacos , Indóis , Longevidade , Camundongos , Neurilemoma/genética , Neurofibromatose 2/metabolismo , Neurofibromina 2/genética , Fosforilação , Piperidinas , Pirimidinas , Células de Schwann/metabolismo , Quinases Ativadas por p21/genéticaRESUMO
Herein, we report the synthesis and antibacterial evaluation of a series of daptomycin lactam-based analogues. As compared with daptomycin, the daptomycin analogue with singly modified lactam has an eightfold increase in its minimum inhibitory concentration (MIC) against methicillin-resistant Staphylococcus aureus. Incorporating effective modifications found in previous daptomycin structure-activity relationship studies to produce lactam-based analogues with multiple modifications did not improve the antibacterial activity of the analogues. Instead, the antibacterial activity was greatly reduced when a rather rigid 4-(phenylethynyl)benzoyl group replaced the flexible n-decanoyl group. The fact that the lactam analogue with the 4-(phenylethynyl)benzoyl group did not exhibit the antibacterial activity comparable to the two respective singly modified analogues showed that the inactivity was probably due to the deviation from the active conformation. This series of lactam analogues may generate insights on the importance of studying the active conformation of daptomycin and how the structural modifications affect the active conformation.
Assuntos
Daptomicina , Staphylococcus aureus Resistente à Meticilina , Daptomicina/farmacologia , Lactamas/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Testes de Sensibilidade MicrobianaRESUMO
Cyclic peptides have been attracting a lot of attention in recent decades, especially in the area of drug discovery, as more and more naturally occurring cyclic peptides with diverse biological activities have been discovered. Chemical synthesis of cyclic peptides is essential when studying their structure-activity relationships. Conventional peptide cyclization methods via direct coupling have inherent limitations, like the susceptibility to epimerization at the C-terminus, poor solubility of fully protected peptide precursors, and low yield caused by oligomerization. In this regard, chemoselective ligation-mediated cyclization methods have emerged as effective strategies for cyclic peptide synthesis. The toolbox for cyclic peptide synthesis has been expanded substantially in the past two decades, allowing more efficient synthesis of cyclic peptides with various scaffolds and modifications. This Review will explore different chemoselective ligation technologies used for cyclic peptide synthesis that generate both native and unnatural peptide linkages. The practical issues and limitations of different methods will be discussed. The advance in cyclic peptide synthesis will benefit the biological and medicinal study of cyclic peptides, an important class of macrocycles with potentials in numerous fields, notably in therapeutics.
Assuntos
Peptídeos Cíclicos/síntese química , Sequência de Aminoácidos , Animais , Bactérias/química , Técnicas de Química Sintética/métodos , Ciclização , Humanos , Plantas/químicaRESUMO
Lysine residues have been considered as a routine conjugating site for protein chemical labeling and modification. The commercially available lysine-labeling agents have several limitations in labeling efficiency, stability, and cost. To pursue alternative protein lysine-labeling strategies, herein, we report the development of an ortho-phthalaldehyde (OPA)-based bifunctional linker suitable for protein chemical labeling and profiling. Among three designed OPA-based bifunctional linkers, OPA-NH-alkyne 5 was proved to be optimal for protein labeling with minimal protein turbidity. We further demonstrated OPA-NH-alkyne 5 was applicable for immediate capture of protein or proteome chemical labeling.
Assuntos
Alcinos/química , Lisina/química , Sondas Moleculares/química , Proteínas/química , o-Ftalaldeído/análogos & derivados , Alcinos/síntese química , Animais , Bactérias/química , Galinhas , Sondas Moleculares/síntese química , o-Ftalaldeído/síntese químicaRESUMO
Chronic hepatitis B virus (HBV) infection has been a serious public health burden worldwide. Current anti-HBV therapies could not eliminate HBV ultimately. Considering the characteristics of HBV, it is impossible to be entirely cured based on current therapies. Therefore, it is urgently needed to develop novel therapeutic agents with new mechanism of action. The dihydroquinolizinone (DHQ) derivatives exhibited potent anti-HBV activity by decreasing HBV DNA and HBsAg level in an obscure mechanism of action. In this study, we have optimized the DHQ scaffold, developed the photoaffinity probe, with which to identify potential binding proteins.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Marcadores de Fotoafinidade/farmacologia , Quinolizinas/farmacologia , Proteínas Virais/análise , Antivirais/síntese química , Cromatografia Líquida , Química Click , Estrutura Molecular , Marcadores de Fotoafinidade/síntese química , Proteoma/análise , Proteoma/química , Proteômica , Quinolizinas/síntese química , Relação Estrutura-Atividade , Espectrometria de Massas em Tandem , Proteínas Virais/químicaRESUMO
A54145B is a calcium-dependent cyclic lipodepsipeptide antibiotic that is active against Gram-positive pathogens. Herein, we report an improved synthetic route toward A54145B in terms of the yield and time required. The key changes include using a pre-assembled minimalist tetradepsipeptide building block to solve the difficult on-resin esterification from our previous synthetic route, and a new macrocyclization site to avoid the peptide self-cleavage problem.
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Megakaryocyte maturation and polyploidization are critical for platelet production; abnormalities in these processes are associated with myeloproliferative disorders, including thrombocytopenia. Megakaryocyte maturation signals through cascades that involve p21-activated kinase (Pak) function; however, the specific role for Pak kinases in megakaryocyte biology remains elusive. Here, we identify Pak2 as an essential effector of megakaryocyte maturation, polyploidization, and proplatelet formation. Genetic deletion of Pak2 in murine bone marrow is associated with macrothrombocytopenia, altered megakaryocyte ultrastructure, increased bone marrow megakaryocyte precursors, and an elevation of mature CD41(+) megakaryocytes, as well as an increased number of polyploid cells. In Pak2(-/-) mice, platelet clearance rate was increased, as was production of newly synthesized, reticulated platelets. In vitro, Pak2(-/-) megakaryocytes demonstrate increased polyploidization associated with alterations in ß1-tubulin expression and organization, decreased proplatelet extensions, and reduced phosphorylation of the endomitosis regulators LIM domain kinase 1, cofilin, and Aurora A/B/C. Together, these data establish a novel role for Pak2 as an important regulator of megakaryopoiesis, polyploidization, and cytoskeletal dynamics in developing megakaryocytes.
Assuntos
Plaquetas/metabolismo , Citoesqueleto/patologia , Megacariócitos/metabolismo , Mitose/genética , Fator de Transcrição PAX2/fisiologia , Trombocitopenia/genética , Trombopoese/fisiologia , Animais , Plaquetas/patologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Citoesqueleto/metabolismo , Megacariócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Microscopia de Fluorescência , Poliploidia , Células-Tronco/metabolismo , Células-Tronco/patologia , Trombocitopenia/patologiaRESUMO
Cysteine-mediated native chemical ligation is a powerful method for protein chemical synthesis. Herein, we report an unprecedentedly mild system (TCEP/NaBH4 or TCEP/LiBEt3 H; TCEP=tris(2-carboxyethyl)phosphine) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation-desulfurization approach. This method, termed P-B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yields, clean conversion, and versatile functionality compatibility with complex peptides/proteins. In addition, this method can be used for incorporating deuterium into the peptides after cysteine desulfurization by running the reaction in D2 O buffer. Moreover, this method enables the clean desulfurization of peptides carrying post-translational modifications, such as phosphorylation and crotonylation. The effectiveness of this method has been demonstrated by the synthesis of the cyclic peptides dichotominâ C and E and synthetic proteins, including ubiquitin, γ-synuclein, and histone H2A.
Assuntos
Proteínas/síntese química , Enxofre/química , Deutério/química , Indicadores e Reagentes/química , Peptídeos/química , Processamento de Proteína Pós-TraducionalRESUMO
In this article, Ser/Thr ligation(on/off) has been realized to enable N-to-C successive peptide ligations using a salicylaldehyde semicarbazone (SAL(off)) group by in situ activation with pyruvic acid of the peptide SAL(off) ester into the peptide salicylaldehyde (SAL(on)) ester. In addition, a peptide with a C-terminal thioester and N-terminal Ser or Thr as the middle peptide segment can undergo one-pot Ser/Thr ligation and native chemical ligation in the N-to-C direction. The utility of this combined ligation strategy in the N-to-C direction has been showcased through the convergent assembly of a human cytokine protein sequence, GlcNAcylated interleukin-25.
Assuntos
Carbono/química , Cisteína/química , Nitrogênio/química , Proteínas/síntese química , Serina/química , Sequência de Aminoácidos , Técnicas de Química Sintética , Interleucina-17/síntese química , Interleucina-17/química , Oligopeptídeos/síntese química , Oligopeptídeos/química , Proteínas/químicaRESUMO
p21-activated kinase-1 (Pak1) is a serine/threonine kinase that plays a key role in mediating antigen-stimulated extracellular calcium influx and degranulation in mast cells. Another isoform in this kinase family, Pak2, is expressed at very high levels in mast cells, but its function is unknown. Here we show that Pak2 loss in murine bone marrow-derived mast cells, unlike loss of Pak1, induces increased antigen-mediated adhesion, degranulation, and cytokine secretion without changes to extracellular calcium influx. This phenotype is associated with an increase in RhoA-GTPase signaling activity to downstream effectors, including myosin light chain and p38(MAPK), and is reversed upon treatment with a Rho-specific inhibitor. Pak2, but not Pak1, negatively regulates RhoA via phosphorylation of the guanine nucleotide exchange factor GEF-H1 at an inhibitory site, leading to increased GEF-H1 microtubule binding and loss of RhoA stimulation. These data suggest that Pak2 plays a unique inhibitory role in mast cell degranulation by down-regulating RhoA via GEF-H1.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Células da Medula Óssea/metabolismo , Cálcio/metabolismo , Adesão Celular , Células Cultivadas , Primers do DNA , Camundongos , Reação em Cadeia da Polimerase , Quinases Ativadas por p21RESUMO
In this work, we developed a novel strategy, prior disulfide bond-mediated Ser/Thr ligation (PD-STL), for the chemical synthesis of peptides and proteins. This approach combines disulfide bond-forming chemistry with Ser/Thr ligation (STL), converting intermolecular STL into intramolecular STL to effectively proceed regardless of concentrations. We demonstrated the effectiveness of PD-STL under high dilution conditions, even for the relatively inert C-terminal proline at the ligation site. Additionally, we applied this method to synthesize the N-terminal cytoplasmic domain (2-104) of caveolin-1 and its Tyr14 phosphorylated form.
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A cell-based transduction inhibition assay (TI) is widely used in clinical trials to detect neutralizing antibody (NAb) titers against recombinant adeno-associated virus (rAAV), one of the most important criteria to exclude patients in gene therapy. Different cell lines are used in cell-based TI because the rAAV transduction efficiencies vary largely among serotypes. A cell line suitable for TI for most serotypes is highly desirable, especially for those with very low transduction efficiencies in vitro such as rAAV8 and rAAV9. Herein, we report an AAVR-HeLa, a stable cell line with overexpressed AAVR, a newly identified receptor for rAAVs, was established for cell-based TIs. The AAVR expression level in AAVR-HeLa cells was approximately 10-fold higher than in HeLa cells, and was stably transfected after twenty three passages. For all AAV serotypes (AAV1-10), except for AAV4, the transduction efficiencies increased significantly in AAVR-HeLa cells. It was demonstrated that the AAVR enhancement of transduction efficiency was only for rAAV and not for lentiviral and adenoviral vectors. According to the minimal multiplicity of infection (MOIs) for the assay, the NAb detection sensitivity increased at least 10 and 20 fold for AAV8 and AAV9, respectively. The seroprevalence of NAbs were investigated at the 1:30 level as a cutoff value using AAVR-HeLa cells. It was shown that the seropositive rate for AAV2 was 87% in serum samples from 99 adults, followed by lower seropositive rates for AAV5 (7%), AAV8 (7%) and AAV9 (1%). Venn diagram analysis showed the presence of cross-reactivity of NAbs to two or three serotypes in 13 samples (13.1%). However, no patient was found to possess NAbs for all the four serotypes. These results demonstrated that the AAVR-HeLa cell line may be utilized to detect the NAbs through cell-based TI assays for most of AAV serotypes.
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MicroRNA-22 (miR-22) was suggested to be important for type 2 diabetes but its functions for this disease remained unclear. Recombinant adeno-associated virus (rAAV)-mediated miR delivery is a powerful approach to study miR functions in vivo, however, the overexpression of miR-22 by rAAV remains challenging because it is one of the most abundant miRs in the liver. In this study, a series of expression cassettes were designed and compared. It was shown that different lengths of primary miR-22 were overexpressed in HEK293 and HeLa cells but the longer ones were more efficiently expressed. miR-22 may be placed in either introns or the 3' UTR of a transgene for efficient overexpression. RNA polymerase III or II promoters were successfully utilized for miR expression but the latter showed higher expression levels in cell lines. Specifically, miR-22 was expressed efficiently together with an EGFP gene. After screening, a liver-specific TTR promoter was chosen to overexpress miR-22 in diabetic mice fed a high-fat diet. It was shown that miR-22 was overexpressed 2-3 folds which improved the insulin sensitivity significantly. The approach utilized in this study to optimize miR overexpression is a powerful tool for the creation of efficient rAAV vectors for the other miRs.
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Overexpression of PAK1, a druggable kinase, is common in several malignancies, and inhibition of PAK1 by small molecules has been shown to impede the growth and survival of such cells. Potent inhibitors of PAKs 1-3 have been described, but clinical development has been hindered by recent findings that PAK2 function is required for normal cardiovascular function in adult mice. A unique allosteric PAK1-selective inhibitor, NVS-PAK1-1, provides a potential path forward, but has modest potency. Here, we report the development of BJG-05-039, a PAK1-selective degrader consisting of NVS-PAK1-1 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon. BJG-05-039 induced selective degradation of PAK1 and displayed enhanced anti-proliferative effects relative to its parent compound in PAK1-dependent, but not PAK2-dependent, cell lines. Our findings suggest that selective PAK1 degradation may confer more potent pharmacological effects compared with catalytic inhibition and highlight the potential advantages of PAK1-targeted degradation.
Assuntos
Dibenzazepinas , Pirrolidinas , Animais , CamundongosRESUMO
Development of safe and efficient vaccines is still necessary to deal with the COVID-19 pandemic. Herein, we reported that yeast-expressed recombinant RBD proteins either from wild-type or Delta SARS-CoV-2 were able to elicit immune responses against SARS-CoV-2 and its variants. The wild-type RBD (wtRBD) protein was overexpressed in Pichia pastoris, and the purified protein was used as the antigen to immunize mice after formulating an aluminium hydroxide (Alum) adjuvant. Three immunization programs with different intervals were compared. It was found that the immunization with an interval of 28 days exhibited the strongest immune response to SARS-CoV-2 than the one with an interval of 14 or 42 days based on binding antibody and the neutralizing antibody (NAb) analyses. The antisera from the mice immunized with wtRBD were able to neutralize the Beta variant with a similar efficiency but the Delta variant with 2~2.5-fold decreased efficiency. However, more NAbs to the Delta variant were produced when the Delta RBD protein was used to immunize mice. Interestingly, the NAbs may cross react with the Omicron variant. To increase the production of NAbs, the adjuvant combination of Alum and CpG oligonucleotides was used. Compared with the Alum adjuvant alone, the NAbs elicited by the combined adjuvants exhibited an approximate 10-fold increase for the Delta and a more than 53-fold increase for the Omicron variant. This study suggested that yeast-derived Delta RBD is a scalable and an effective vaccine candidate for SARS-CoV-2 and its variants.
Assuntos
COVID-19 , Vacinas Virais , Camundongos , Humanos , Animais , SARS-CoV-2 , Saccharomyces cerevisiae , Vacinas contra COVID-19 , Pandemias , Camundongos Endogâmicos BALB C , COVID-19/prevenção & controle , Adjuvantes Imunológicos , Proteínas Recombinantes , ImunidadeRESUMO
BACKGROUND: Recently, through comprehensive medicinal chemistry efforts, we have found a new daptomycin analogue, termed kynomycin, showing enhanced activity against both methicillin-resistant S. aureus and vancomycin-resistant Enterococcus in vitro and in vivo, with improved pharmacokinetics and lower cytotoxicity than daptomycin. METHODS: In this study we compared the physicochemical properties of kynomycin with those of daptomycin from an atomic perspective by using Nuclear Magnetic Resonance spectroscopy and Molecular Dynamics simulations. RESULTS AND CONCLUSION: We observed that kynurenine methylation changes daptomycin's key physicochemical properties; its calcium dependent oligomerization efficiency is improved and the modified kynurenine strengths contacts with the lipid tail and tryptophan residues. In addition, it is observed that, compared to daptomycin, kynomycin tetramer is more stable and binds stronger to calcium. The combined experiments provide key clues for the improved antibacterial activity of kynomycin. GENERAL SIGNIFICANCE: We expect that this approach will help study the calcium binding and oligomerization features of new calcium dependent peptide antibiotics.
Assuntos
Cálcio/química , Cálcio/metabolismo , Daptomicina/química , Daptomicina/metabolismo , Simulação de Dinâmica Molecular , Antibacterianos/química , Antibacterianos/metabolismo , Depsipeptídeos/química , Depsipeptídeos/metabolismo , Lipopeptídeos/química , Lipopeptídeos/metabolismoRESUMO
Daptomycin is effective in treating infections caused by antibiotic-resistant Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and vancomycin-resistant S. aureus (VRSA). Due to its distinct mechanism of action toward multidrug-resistant bacteria, daptomycin provides an attractive structural motif to generate new daptomycin-based antibiotics to combat the problem of bacterial resistance. In this study, we used the total synthesis method to produce daptomycin analogues with a variety in terms of types and sites of modifications. Five classes of daptomycin analogues were synthesized, and the antimicrobial activities of the analogues were analyzed by several biological assays. From this study, we established a comprehensive structure-activity relationship of daptomycin which will lay the foundation for the further development of daptomycin-based antibiotics.
RESUMO
Increased usage of daptomycin to treat infections caused by Gram-positive bacterial pathogens has resulted in emergence of resistant mutants. In a search for more effective daptomycin analogues through medicinal chemistry studies, we found that methylation at the nonproteinogenic amino acid kynurenine in daptomycin could result in significant enhancement of antibacterial activity. Termed "kynomycin," this new antibiotic exhibits higher antibacterial activity than daptomycin and is able to eradicate methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) strains, including daptomycin-resistant strains. The improved antimicrobial activity of kynomycin was demonstrated in in vitro time-killing assay, in vivo wax worm model, and different mouse infection models. The increased antibacterial activity, improved pharmacokinetics, and lower cytotoxicity of kynomycin, compared to daptomycin, showed the promise of the future design and development of next-generation daptomycin-based antibiotics.
Assuntos
Antibacterianos/uso terapêutico , Depsipeptídeos/uso terapêutico , Lipopeptídeos/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Animais , Antibacterianos/síntese química , Antibacterianos/farmacocinética , Antibacterianos/toxicidade , Permeabilidade da Membrana Celular/efeitos dos fármacos , Daptomicina/química , Daptomicina/uso terapêutico , Depsipeptídeos/síntese química , Depsipeptídeos/farmacocinética , Depsipeptídeos/toxicidade , Farmacorresistência Bacteriana/efeitos dos fármacos , Enterococcus/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Lepidópteros/efeitos dos fármacos , Lepidópteros/microbiologia , Lipopeptídeos/síntese química , Lipopeptídeos/farmacocinética , Lipopeptídeos/toxicidade , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Metilação , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Testes de Sensibilidade MicrobianaRESUMO
Gene expression studies are important for revealing gene functions putatively involved in biological processes. We were interested in identifying differentially expressed genes during leaf development in rice. We combined the RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and dot blot hybridization methods to screen a rice leaf primordium cDNA library. Three developmental stages during vegetative growth were examined. The cDNA clones showing different hybridization patterns were further analyzed and verified. Here we demonstrate that the combination of RAP-PCR and dot blot hybridization could provide an efficient and relatively low-cost cDNA library screening approach to discover genes not previously known to be associated with leaf development in rice. We believe that the findings described here will help to elucidate the molecular mechanism(s) underlying the developmental processes of rice leaf.