RESUMO
Catalytic metallopeptides that target the membrane-associated sortase A transpeptidase have been developed and evaluated as irreversible inactivators of SrtA∆N59 (sortase A, lacking the initial membrane-binding domain). The copper-binding GGH tripeptide ATCUN motif was linked to amidated forms of the cell wall sorting signal, LPET and LPETG, as sortase-targeting moieties. The resulting metallopeptides were used to determine half maximal inhibitory concentrations (IC50) and rate constants for time-dependent sortase A inactivation. Michaelis-Menten behavior was observed for the catalytic metallopeptides, and k(cat), K(M) and k(cat)/K(M) parameters were obtained as 0.080 ± 0.002 min⻹, 23 ± 2 µM and 0.0035 ± 0.0003 µM⻹ min⻹, respectively. Concentration-dependent inhibition of SrtA∆N59 by the metallopeptides revealed IC50 values ranging from 570 to 700 µM, while Cu-GGH, which lacked a targeting motif, had no measurable IC50 value (>2,000 µM). Time-dependent inactivation of SrtA revealed a range of catalytic activities, with Cu-GGHGLPETG-NH2 demonstrating the fastest rate of inactivation in the presence of ascorbate and hydrogen peroxide coreactants. The active site of the enzyme comprises residues Cys-184, Arg-197 and His-120. LC-MS/MS analysis of the reaction products demonstrated modification of Cys-184 to cysteine sulfonic acid (+48 amu). Results obtained from a DTNB assay support oxidation of the Cys-184 residue. LC-MS/MS also suggested oxidation of the Arg-197 containing peptide. 2D NMR analysis was performed to assess the possible oxidation of His-120, however, none was observed. These compounds possess the potential for irreversible inactivation of SrtA through oxidative modification of essential residues required for substrate binding.
Assuntos
Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Cobre/química , Inibidores Enzimáticos/farmacologia , Níquel/química , Oligopeptídeos/química , Compostos Organometálicos/farmacologia , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Relação Estrutura-AtividadeRESUMO
Porcine circovirus type 2d (PCV2d) is becoming the predominant PCV genotype and considerably affects the global pig industry. Nevertheless, currently, no commercial PCV2d vaccine is available. Preventing and controlling the disease caused by PCV2d is therefore based on other genotype-based vaccines. However, their production platforms are laborious, limited in expression level, and relatively expensive for veterinary applications. To address these challenges, we have developed a simple and cost-efficient platform for a novel PCV2d vaccine production process, using fed-batch E. coli fermentation followed by cell disruption and filtration, and a single purification step via cation exchange chromatography. The process was developed at bench scale and then pilot scale, where the PCV2d subunit protein yield was approximately 0.93 g/L fermentation volume in a short production time. Moreover, we have successfully implemented this production process at two different sites, in Southeast Asia and Europe. This demonstrates transferability and the high potential for successful industrial production.
RESUMO
A series of compounds that target reactive transition-metal chelates to somatic angiotensin converting enzyme (sACE-1) have been synthesized. Half-maximal inhibitory concentrations (IC(50)) and rate constants for both inactivation and cleavage of full-length sACE-1 have been determined and evaluated in terms of metal chelate size, charge, reduction potential, coordination unsaturation, and coreactant selectivity. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and tripeptide GGH were linked to the lysine side chain of lisinopril by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride/N-hydroxysuccinimide coupling. The resulting amide-linked chelate-lisinopril (EDTA-lisinopril, NTA-lisinopril, DOTA-lisinopril, and GGH-lisinopril) conjugates were used to form coordination complexes with iron, cobalt, nickel, and copper, such that lisinopril could mediate localization of the reactive metal chelates to sACE-1. ACE activity was assayed by monitoring cleavage of the fluorogenic substrate Mca-RPPGFSAFK(Dnp)-OH, a derivative of bradykinin, following preincubation with metal chelate-lisinopril compounds. Concentration-dependent inhibition of sACE-1 by metal chelate-lisinopril complexes revealed IC(50) values ranging from 44 to 4500 nM for Ni-NTA-lisinopril and Ni-DOTA-lisinopril, respectively, versus 1.9 nM for lisinopril. Stronger inhibition was correlated with smaller size and lower negative charge of the attached metal chelates. Time-dependent inactivation of sACE-1 by metal chelate-lisinopril complexes revealed a remarkable range of catalytic activities, with second-order rate constants as high as 150,000 M(-1) min(-1) (Cu-GGH-lisinopril), while catalyst-mediated cleavage of sACE-1 typically occurred at much lower rates, indicating that inactivation arose primarily from side chain modification. Optimal inactivation of sACE-1 was observed when the reduction potential for the metal center was poised near 1000 mV, reflecting the difficulty of protein oxidation. This class of metal chelate-lisinopril complexes possesses a range of high-affinity binding to ACE, introduces the advantage of irreversible catalytic turnover, and marks an important step toward the development of multiple-turnover drugs for selective inactivation of sACE-1.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Quelantes/química , Lisinopril/análogos & derivados , Peptidil Dipeptidase A/metabolismo , Elementos de Transição/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Catálise , Quelantes/farmacologia , Humanos , Lisinopril/farmacologia , Modelos Moleculares , Peptidil Dipeptidase A/químicaRESUMO
Amphiphilic chitosan, bPalm-CS-HTAP, having N-(2-((2,3-bis(palmitoyloxy)propyl)amino)-2-oxoethyl) (bPalm) groups as double hydrophobic tails and O-[(2-hydroxyl-3-trimethylammonium)] propyl (HTAP) groups as hydrophilic heads was synthesized and evaluated for its self-assembly properties and potential as a gene carrier. The degree of bis-palmitoyl group substitution (DS bPalm) and the degree of quaternization (DQ) were approximately 2 and 56%, respectively. bPalm-CS-HTAP was found to assemble into nanosized spherical particles with a hydrodynamic diameter (D H) of 265.5 ± 7.40 nm (PDI = 0.5) and a surface charge potential of 40.1 ± 0.04 mV. bPalm-CS-HTAP condensed the plasmid pVAX1.CoV2RBDme completely at a bPalm-CS-HTAP:pDNA ratio of 2:1. The self-assembled bPalm-CS-HTAP/pDNA complexes could enter HEK 293A and CHO cells and enabled gene expression at negligible cytotoxicity compared to commercial PEI (20 kDa). These results suggested that bPalm-CS-HTAP can be used as a promising nonviral gene carrier.
RESUMO
DNA vaccines, the third generation of vaccines, are a promising therapeutic option for many diseases as they offer the customization of their ability on protection and treatment with high stability. The production of DNA vaccines is considered rapid and less complicated compared to others such as mRNA vaccines, viral vaccines, or subunit protein vaccines. However, the main issue for DNA vaccines is how to produce the active DNA, a supercoiled isoform, to comply with the regulations. Our work therefore focuses on gaining a process understanding of the purification step which processes parameters that have impacts on the critical quality attribute (CQA), supercoiled DNA and performance attribute (PA), and step yield. Herein, pVax1/lacZ was used as a model. The process parameters of interest were sample application flow rates and salt concentration at washing step and at elution step in the hydrophobic interaction chromatography (HIC). Using a Design of Experiment (DoE) with central composite face centered (CCF) approach, 14 experiments plus four additional runs at the center points were created. The response data was used to establish regression predictive models and simulation was conducted in 10,000 runs to provide tolerance intervals of these CQA and PA. The approach of this process understanding can be applied for Quality by Design (QbD) on other DNA vaccines and on a larger production scale as well.
RESUMO
Plasmid DNA is a vital biological tool for molecular cloning and transgene expression of recombinant proteins; however, decades ago, it has become an exceptionally appealing as a potential biopharmaceutical product as genetic immunization for animal and human use. The demand for large-quantity production of DNA vaccines also increases. Thus, we, herein, presented a systematic approach for process characterization of fed-batch Escherichia coli DH5α fermentation producing a porcine DNA vaccine. Design of Experiments (DoE) was employed to determine process parameters that have impacts on a critical quality attribute of the product, which is the active form of plasmid DNA referred as supercoiled plasmid DNA content, as well as the performance attributes, which are volumetric yield and specific yield from fermentation. The parameters of interest were temperature, pH, dissolved oxygen, cultivation time, and feed rate. Using the definitive-screening design, there were 16 runs, including 3 additional center points to create the predictive model, which then was used to simulate the operational ranges for capability analysis.
RESUMO
A new paradigm for drug activity is presented, which includes both recognition and subsequent irreversible inactivation of therapeutic targets. Application to both RNA and protein biomolecules has been demonstrated. In contrast to RNA targets that are subject to strand scission chemistry mediated by ribose H-atom abstraction, proteins appear to be inactivated either through oxidative damage to amino acid side chains around the enzyme active site, or by backbone hydrolysis.
Assuntos
Enzimas/química , Metais/química , Peptídeo Hidrolases/química , RNA/química , Sítios de Ligação , Desenho de Fármacos , HidróliseRESUMO
The N- and C-terminal domains of human somatic angiotensin I converting enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates was tested for both reversible binding and irreversible catalytic inactivation of each domain of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and catalytic factors (double-filter effect).