RESUMO
Natural killer (NK) cells are key players in human innate immunity. Cell engager antibody formats that recruit and activate NK cells more effectively have emerged as a promising immunotherapy approach to target cancer cells through more effective antibody-dependent cell-mediated cytotoxicity (ADCC). Monoclonal antibody drugs with ADCC activity have shown clinical benefit and improved outcomes for patients with certain types of cancer. CD16a, a Fc gamma III receptor, is the major component that is responsible for the ADCC activity of NK cells. Screening AvantGen's yeast displayed human antibody libraries led to the isolation of 2 antibody clones, #1A2 and #2-2A2, that selectively recognize both isoforms (F and V) of CD16a on primary NK cells with high affinity, yet minimally (#1A2) or do not (#2-2A2) cross-react with both allelotypes of CD16b (NA1 and NA2) expressed by neutrophils. Epitope mapping studies revealed that they bind to an epitope dependent on residue Y158 of CD16a, since mutation of Y158 to the corresponding CD16b residue H158 completely abolishes binding to CD16a. When formatted as bispecific antibodies targeting CD16a and a tumor-associated antigen (TAA, e.g. CD19), they exhibit specific binding to NK cells and induce potent NK cell activation upon encountering tumor cells, resulting in effective tumor cell killing. Notably, these bispecific antibody engagers stimulate NK cell cytokine release during co-culture with target cells, resulting in target cell cytotoxicity. These anti-CD16a antibody clones are promising candidates for combination with any TAA of interest, offering the potential for novel NK cell engager-based cancer therapeutics that are minimally affected by the high concentrations of human IgG in the circulation.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Células Matadoras Naturais , Receptores de IgG , Humanos , Células Matadoras Naturais/imunologia , Receptores de IgG/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Mapeamento de Epitopos/métodos , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/tratamento farmacológicoRESUMO
Antibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the 'opioid epidemic.' We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.
Assuntos
Anticorpos/farmacologia , Especificidade de Anticorpos , Ensaios de Triagem em Larga Escala , Proteína Quinase C/antagonistas & inibidores , Receptores Opioides mu/antagonistas & inibidores , Sinapses/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Animais , Anticorpos/imunologia , Linhagem Celular Tumoral , Ativação Enzimática , Fentanila/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Fosforilação , Proteína Quinase C/imunologia , Proteína Quinase C/metabolismo , Coelhos , Receptores Opioides mu/imunologia , Receptores Opioides mu/metabolismo , Transdução de Sinais , Sinapses/imunologia , Sinapses/metabolismo , Fatores de TempoRESUMO
The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro. Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Tratamento Farmacológico da COVID-19 , COVID-19 , Imunoglobulina G , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , COVID-19/sangue , COVID-19/imunologia , Chlorocebus aethiops , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Masculino , Mesocricetus , Índice de Gravidade de Doença , Células Vero , Carga Viral/efeitos dos fármacos , Carga Viral/imunologiaRESUMO
The emergence of COVID-19 has led to a pandemic that has caused millions of cases of disease, variable morbidity and hundreds of thousands of deaths. Currently, only remdesivir and dexamethasone have demonstrated limited efficacy, only slightly reducing disease burden, thus novel approaches for clinical management of COVID-19 are needed. We identified a panel of human monoclonal antibody clones from a yeast display library with specificity to the SARS-CoV-2 spike protein receptor binding domain that neutralized the virus in vitro . Administration of the lead antibody clone to Syrian hamsters challenged with SARS-CoV-2 significantly reduced viral load and histopathology score in the lungs. Moreover, the antibody interrupted monocyte infiltration into the lungs, which may have contributed to the reduction of disease severity by limiting immunopathological exacerbation. The use of this antibody could provide an important therapy for treatment of COVID-19 patients.
RESUMO
A series of peptidic fluorogenic substrates were synthesized to develop a flow cytometry assay (FACS) to monitor the proteolytic activity of cathepsin C in live cells. Of the 16 substrates tested, (NH(2)-aminobutyric-homophenylalanine)(2)-rhodamine demonstrated the best reactivity and selectivity profile in the FACS assay using the B721 human B-lymphoblastoid cell line. The resulting FACS assay was validated through correlation of the IC(50) values with a competitive radiolabeling assay against a series of small molecule inhibitors of cathepsin C.
Assuntos
Catepsina C/metabolismo , Corantes Fluorescentes/química , Rodaminas/química , Catepsina C/antagonistas & inibidores , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Marcação por Isótopo , Radioisótopos/química , Rodaminas/síntese química , Especificidade por SubstratoRESUMO
PCSK9 (proprotein convertase subtilisin/kexin 9) is a secreted serine protease that regulates cholesterol homoeostasis by inducing post-translational degradation of hepatic LDL-R [LDL (low-density lipoprotein) receptor]. Intramolecular autocatalytic processing of the PCSK9 zymogen in the endoplasmic reticulum results in a tightly associated complex between the prodomain and the catalytic domain. Although the autocatalytic processing event is required for proper secretion of PCSK9, the requirement of proteolytic activity in the regulation of LDL-R is currently unknown. Co-expression of the prodomain and the catalytic domain in trans allowed for production of a catalytically inactive secreted form of PCSK9. This catalytically inactive PCSK9 was characterized and shown to be functionally equivalent to the wild-type protein in lowering cellular LDL uptake and LDL-R levels. These findings suggest that, apart from autocatalytic processing, the protease activity of PCSK9 is not necessary for LDL-R regulation.
Assuntos
Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Linhagem Celular , Humanos , Mutação/genética , Serina/genética , Serina/metabolismo , Serina Endopeptidases/genéticaRESUMO
A 6-oxa-1-aza-bicyclo[3.2.1]octan-7-one system inhibits the proteolytic activity of several cysteine proteases belonging to the papain family. In vitro mechanistic studies and in silico calculations suggest that the minimal pi-overlap between the bridgehead nitrogen and the carbonyl leads to a considerable weakening of the urethane system, making it susceptible to nucleophilic attack from the active site thiol group. The resulting covalent adduct is slowly hydrolyzed, releasing the hydroxypiperidine product of the inhibitor. Synthesis and testing of a set of analogs led to variable protease subtype selectivities ranging from micromolar to nanomolar potencies.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carbamatos/síntese química , Carbamatos/farmacologia , Catepsinas/antagonistas & inibidores , Sítios de Ligação , Biologia Computacional , Elétrons , Modelos Moleculares , Papaína/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Peptidic, non-covalent inhibitors of lysosomal cysteine protease cathepsin S (1 and 2) were investigated due to low oral bioavailability, leading to an improved series of peptidomimetic inhibitors. Utilizing phenyl succinamides as the P2 residue increased the oral exposure of this lead series of compounds, while retaining selective inhibition of the cathepsin S isoform. Concurrent investigation of the P1 and P2 subsites resulted in the discovery of several potent and selective inhibitors of cathepsin S with good pharmacokinetic properties due to the elimination of saturated aliphatic P2 residues.
Assuntos
Amidas/síntese química , Catepsinas/antagonistas & inibidores , Inibidores de Proteases/síntese química , Amidas/química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Desenho de Fármacos , Masculino , Estrutura Molecular , Inibidores de Proteases/química , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Ratos , Ratos Wistar , Relação Estrutura-Atividade , SuccinatosRESUMO
We report a novel series of noncovalent inhibitors of cathepsin S. The synthesis of the peptidomimetic scaffold is described and structure-activity relationships of P3, P1, and P1' subunits are discussed. Lead optimization to a non-peptidic scaffold has resulted in a new class of potent, highly selective, and orally bioavailable cathepsin S inhibitors.
Assuntos
Carbamatos/síntese química , Carbamatos/farmacologia , Catepsinas/antagonistas & inibidores , Oligopeptídeos/síntese química , Inibidores de Proteases/síntese química , Administração Oral , Animais , Disponibilidade Biológica , Carbamatos/farmacocinética , Humanos , Masculino , Mimetismo Molecular , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
A systematic study of anilines led to the discovery of a metabolically robust fluoroindoline replacement for the alkoxy aniline toxicophore in 1. Investigations of the P1 pocket resulted in the discovery of a wide tolerance of functionality leading to the discovery of 11 as a potent and selective inhibitor of cathepsin S.
Assuntos
Amidas/farmacologia , Catepsinas/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Desenho de Fármacos , Amidas/síntese química , Amidas/química , Aminação , Derivados de Benzeno/síntese química , Derivados de Benzeno/química , Derivados de Benzeno/farmacologia , Sítios de Ligação , Catepsina K , Catepsina L , Catepsinas/química , Catepsinas/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Etanol/química , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.
Assuntos
Amidas/síntese química , Amidas/farmacologia , Catepsinas/antagonistas & inibidores , Administração Oral , Amidas/farmacocinética , Animais , Sítios de Ligação , Disponibilidade Biológica , Cristalografia por Raios X , Éteres Cíclicos/síntese química , Éteres Cíclicos/farmacologia , Humanos , Masculino , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Ratos , Ratos Wistar , Relação Estrutura-Atividade , ZincoRESUMO
A series of N(alpha)-2-benzoxazolyl-alpha-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure-activity relationships including strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound 3 bound to the active site of cathepsin S is also reported.
Assuntos
Amidas/farmacologia , Catepsinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/farmacologia , Amidas/química , Animais , Catepsinas/química , Catepsinas/genética , Catepsinas/fisiologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Compostos Heterocíclicos/química , Camundongos , Camundongos Knockout , Modelos Moleculares , RatosRESUMO
A strategy was developed to determine the prime and non-prime substrate specificity of serine, threonine and cysteine proteases. ACC positional scanning technology was employed to determine the P4-P1 non-prime site substrate specificity. The data was used to synthesize biased donor-quencher positional scanning libraries to profile the P1'-P4' prime site substrate specificity. Directed sorting using the Irori Nanokan system allowed for the archiving of multiple P1'-P4' positional scanning libraries. From these libraries focused donor-quencher libraries incorporating P4-P1 data for each protease under study could be rapidly prepared. The profiling of thrombin and caspase-3 P4-P4' substrate specificity, comparison of the library specificity data to single substrates, and the analysis of physiological cleavage sites are described.
Assuntos
Caspases/metabolismo , Técnicas de Química Combinatória , Cumarínicos/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Caspase 3 , Simulação por Computador , Cumarínicos/química , Corantes Fluorescentes/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.
Assuntos
Vírus da Dengue/genética , Perfilação da Expressão Gênica , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Vírus da Dengue/classificação , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Biblioteca de Peptídeos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Sorotipagem , Especificidade por SubstratoRESUMO
A series of Nalpha-acyl-alpha-amino acid-(arylaminoethyl)amides were found to be potent and noncovalent cathepsin S inhibitors. Compound 20 possessed high cathepsin S affinity (Ki=3.3 nM) and showed excellent selectivity over cathepsin K, L, F, and V. Molecular modeling, design, synthesis, and in vitro activity are described.