RESUMO
Reversed-phase liquid chromatographic mass spectrometry (rpLC-MS) is a universal, platformed, and essential analytical technique within pharmaceutical and biopharmaceutical research. Typical rpLC method gradient times can range from 5 to 20 min. As monoclonal antibody (mAb) therapies continue to evolve and bispecific antibodies (BsAbs) become more established, research stage engineering panels will clearly evolve in size. Therefore, high-throughput (HT) MS and automated deconvolution methods are key for success. Additionally, newer therapeutics such as bispecific T-cell engagers and nucleic acid-based modalities will also require MS characterization. Herein, we present a modality and target agnostic HT solid-phase extraction (SPE) MS method that affords the analysis of a 96-well plate in 41.4 min, compared to the traditional rpLC-MS method that would typically take 14.4 h. The described method can accurately determine the molecular weights for monodispersed and highly polydispersed biotherapeutic species and membrane proteins; determine levels of glycosylation, glycation, and formylation; detect levels of chain mispairing; and determine accurate drug-to-antibody ratio values.
Assuntos
Cromatografia de Fase Reversa , Extração em Fase Sólida , Espectrometria de Massas/métodos , Cromatografia de Fase Reversa/métodos , Peso Molecular , Anticorpos Monoclonais/químicaRESUMO
Inhibition of the pituitary adenylate cyclase 1 receptor (PAC1R) is a novel mechanism that could be used for abortive treatment of acute migraine. Our research began with comparative analysis of known PAC1R ligand scaffolds, PACAP38 and Maxadilan, which resulted in the selection of des(24-42) Maxadilan, 6, as a starting point. C-terminal modifications of 6 improved the peptide metabolic stability in vitro and in vivo. SAR investigations identified synergistic combinations of amino acid replacements that significantly increased the in vitro PAC1R inhibitory activity of the analogs to the pM IC90 range. Our modifications further enabled deletion of up to six residues without impacting potency, thus improving peptide ligand binding efficiency. Analogs 17 and 18 exhibited robust in vivo efficacy in the rat Maxadilan-induced increase in blood flow (MIIBF) pharmacodynamic model at 0.3 mg/kg subcutaneous dosing. The first cocrystal structure of a PAC1R antagonist peptide (18) with PAC1R extracellular domain is reported.
Assuntos
Circulação Sanguínea/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/antagonistas & inibidores , Animais , Humanos , Proteínas de Insetos/farmacologia , Masculino , Camundongos , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/metabolismo , Transtornos de Enxaqueca/fisiopatologia , Simulação de Acoplamento Molecular , Peptídeos/farmacocinética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Vasodilatadores/farmacologiaRESUMO
We report a metal free synthetic hydrogel copolymer with affinity and selectivity for His6-tagged peptides and proteins. Small libraries of copolymers incorporating charged and hydrophobic functional groups were screened by an iterative process for His6 peptide affinity. The monomer selection was guided by interactions found in the crystal structure of an anti-His tag antibody-His6 peptide antigen complex. Synthetic copolymers incorporating a phenylalanine-derived monomer were found to exhibit strong affinity for both His6-containing peptides and proteins. The proximity of both aromatic and negatively charged functional groups were important factors for the His6 affinity of hydrogel copolymers. His6 affinity was not compromised by the presence of enzyme cleavage sequences. The His6-copolymer interactions are pH sensitive: the copolymer selectively captured His6 peptides at pH 7.8 while the interactions were substantially weakened at pH 8.6. This provided mild conditions for releasing His6-tagged proteins from the copolymer. Finally, a synthetic copolymer coated chromatographic medium was prepared and applied to the purification of a His6-tagged protein from an E. coli expression system. The results establish that a synthetic copolymer-based affinity medium can function as an effective alternative to immobilized metal ion columns for the purification of His6-tagged proteins.
Assuntos
Escherichia coli , Polímeros , Cromatografia de Afinidade , Escherichia coli/genética , Metais , Proteínas , Proteínas RecombinantesRESUMO
The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = -0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies. SIGNIFICANCE STATEMENT: Although multiple in vitro and in silico tools are available in small-molecule drug discovery, pharmacokinetic characterization of protein therapeutics is still highly dependent upon the use of in vivo studies in preclinical species. The current work demonstrates the combined use of cryopreserved hepatocytes, hydrophobic interaction chromatography, and neonatal Fc receptor binding to characterize a series of Nav1.7 peptide-antibody conjugates prior to conducting in vivo studies, thus providing a means to rapidly evaluate novel protein therapeutic platforms while concomitantly reducing the number of in vivo studies conducted in preclinical species.
Assuntos
Dor Crônica/tratamento farmacológico , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoconjugados/farmacocinética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Receptores Fc/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Administração Intravenosa , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Criopreservação , Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos , Antígenos de Histocompatibilidade Classe I/genética , Imunoconjugados/administração & dosagem , Macaca fascicularis , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Knockout , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Receptores Fc/genética , Distribuição Tecidual , Bloqueadores do Canal de Sódio Disparado por Voltagem/administração & dosagemRESUMO
Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.
Assuntos
Imunoconjugados , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/química , Venenos de Aranha/química , Bloqueadores do Canal de Sódio Disparado por Voltagem , Animais , Anticorpos/química , Descoberta de Drogas , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Masculino , Camundongos , Terapia de Alvo Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/imunologia , Peptídeos/farmacocinética , Venenos de Aranha/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinéticaRESUMO
Inhibitors of the voltage-gated sodium channel NaV1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified NaV1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived NaV1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic NaV1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic NaV1.7 inhibitors.
Assuntos
Descoberta de Drogas , Histamina/efeitos adversos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Prurido/tratamento farmacológico , Venenos de Aranha/química , Animais , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/química , Peptídeos/farmacocinética , Peptídeos/uso terapêutico , Conformação Proteica , Dobramento de Proteína , Prurido/induzido quimicamente , Relação Estrutura-Atividade , Distribuição Tecidual , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
To realize the medicinal potential of peptide toxins, naturally occurring disulfide-rich peptides, as ion channel antagonists, more efficient pharmaceutical optimization technologies must be developed. Here, we show that the therapeutic properties of multiple cysteine toxin peptides can be rapidly and substantially improved by combining direct chemical strategies with high-throughput electrophysiology. We applied whole-molecule, brute-force, structure-activity analoging to ShK, a peptide toxin from the sea anemone Stichodactyla helianthus that inhibits the voltage-gated potassium ion channel Kv1.3, to effectively discover critical structural changes for 15× selectivity against the closely related neuronal ion channel Kv1.1. Subsequent site-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000× over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmacological potential of the optimized toxin peptide was demonstrated by potent and sustained inhibition of cytokine secretion from T cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys.
Assuntos
Venenos de Cnidários/química , Canal de Potássio Kv1.3/antagonistas & inibidores , Peptídeos/química , Polietilenoglicóis/química , Animais , Células CHO , Venenos de Cnidários/farmacocinética , Venenos de Cnidários/farmacologia , Cricetulus , Cristalografia por Raios X , Cães , Células HEK293 , Humanos , Interferon gama/sangue , Interferon gama/metabolismo , Interleucina-17/sangue , Interleucina-17/metabolismo , Interleucina-2/sangue , Interleucina-2/metabolismo , Canal de Potássio Kv1.1/antagonistas & inibidores , Macaca fascicularis , Masculino , Camundongos , Simulação de Acoplamento Molecular , Técnicas de Patch-Clamp , Peptídeos/farmacocinética , Peptídeos/farmacologia , Ratos Sprague-Dawley , Especificidade da Espécie , Estereoisomerismo , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Since the advent of solid-phase peptide synthesis (SPPS) in the late 1950s, numerous advancements in the underlying chemistry (i.e., orthogonal protection strategy, coupling reagents, and solid support matrices) have greatly improved the efficiency of the technique. More recently, application of microwave radiation to SPPS has been found to reduce reaction time and/or increase the initial purity of synthetic peptide products. In this protocol, conditions are described to accomplish rapid peptide coupling and 9-fluorenylmethoxycarbonyl (Fmoc) removal reactions under temperature-controlled conditions in either a manual or automated synthesis format using a microwave reactor. These microwave-assisted peptide synthesis procedures have been used to rapidly prepare a "difficult" peptide sequence from the acyl carrier protein, ACP(65-74), in less than 3 h and the reduced, linear precursor to human hepcidin, in high initial purity.
Assuntos
Técnicas de Química Combinatória/métodos , Micro-Ondas , Peptídeos/síntese química , Proteína de Transporte de Acila/síntese química , Proteína de Transporte de Acila/química , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Técnicas de Química Combinatória/economia , Técnicas de Química Combinatória/instrumentação , Fluorenos , Hepcidinas , Humanos , Dados de Sequência Molecular , Peptídeos/químicaRESUMO
Calcitonin gene-related peptide (CGRP) is a 37-residue neuropeptide that can be converted to a CGRP(1) receptor antagonist by the truncation of its first seven residues. CGRP(8-37), 1, has a CGRP(1) receptor K(i) = 3.2 nM but is rapidly degraded in human plasma (t(1/2) = 20 min). As part of an effort to identify a prolonged in vivo circulating CGRP peptide antagonist, we found that the substitution of multiple residues in the CGRP peptide increased CGRP(1) receptor affinity >50-fold. Ac-Trp-[Arg(24),Lys(25),Asp(31),Pro(34),Phe(35)]CGRP(8-37)-NH(2), 5 (K(i) = 0.06 nM) had the highest CGRP(1) receptor affinity. Using complimentary in vitro and in vivo metabolic studies, we iteratively identified degradation sites and prepared high affinity analogues with significantly improved plasma stability. Ac-Trp-[Cit(11,18),hArg(24),Lys(25),2-Nal(27,37),Asp(31),Oic(29,34),Phe(35)]CGRP(8-37)-NH(2), 32 (K(i) = 3.3 nM), had significantly increased (>100-fold) stability over 1 or 5, with a cynomolgus monkey and human in vitro plasma half-life of 38 and 68 h, respectively.
Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Química Farmacêutica/métodos , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Cinética , Macaca fascicularis , Masculino , Conformação Molecular , Dados de Sequência Molecular , Ligação ProteicaRESUMO
A series of conformationally constrained derivatives of glucagon-like peptide-1 (GLP-1) were designed and evaluated. By use of [Gly (8)]GLP-1(7-37)-NH2 (2) peptide as a starting point, 17 cyclic derivatives possessing i to i + 4, i to i + 5, or i to i + 7 side chain to side chain lactam bridges from positions 18 to 30 were prepared. The effect of a helix-promoting alpha-amino-isobutyric acid (Aib) substitution at position 22 was also evaluated. The introduction of i to i + 4 glutamic acid-lysine lactam constraints in c[Glu (18)-Lys (22)][Gly (8)]GLP-1(7-37)-NH2 (6), c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-NH2 (10), and c[Glu (23)-Lys (27)][Gly (8)]GLP-1(7-37)-NH2 (11) resulted in potent functional activity and receptor affinities comparable to native GLP-1. Selected GLP-1 peptides were chemoselectively PEGylated in order to prolong their in vivo activity. PEGylated peptides [Gly (8),Aib (22)]GLP-1(7-37)-Cys ((PEG))-Ala-NH2 (23) and c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-Cys ((PEG))-Ser-Gly-NH2 (24) retained picomolar functional potency and avid receptor binding properties. Importantly, PEGylated GLP-1 peptide 23 exhibited sustained in vivo efficacy with respect to blood glucose reduction and decreased body weight for several days in nonhuman primates.