RESUMEN
CTLA4-Ig/abatacept dampens activation of naive T cells by blocking costimulation via CD28. It is an approved drug for rheumatoid arthritis but failed to deliver efficacy in a number of other autoimmune diseases. One explanation is that activated T cells rely less on CD28 signaling and use alternate coreceptors for effector function. ICOS is critical for activation of T-dependent humoral immune responses, which drives pathophysiology of IgG-mediated autoimmune diseases. In this study, we asked whether CD28 and ICOS play nonredundant roles for maintenance of T-dependent responses in mouse models. Using a hapten-protein immunization model, we show that during an ongoing germinal center response, combination treatment with CTLA4-Ig and ICOS ligand (ICOSL) blocking Ab completely dissolves ongoing germinal center responses, whereas single agents show only partial activity. Next, we took two approaches to engineer a therapeutic molecule that blocks both pathways. First, we engineered CTLA4-Ig to enhance binding to ICOSL while retaining affinity to CD80/CD86. Using a library approach, binding affinity of CTLA4-Ig to human ICOSL was increased significantly from undetectable to 15-42 nM; however, the affinity was still insufficient to completely block binding of ICOSL to ICOS. Second, we designed a bispecific costimulation inhibitor with high-affinity CTLA4 extracellular domains fused to anti-ICOSL Ab termed bifunctional costimulation inhibitor. With this bispecific approach, we achieved complete inhibition of CD80 and CD86 binding to CD28 as well as ICOS binding to ICOSL. Such bispecific molecules may provide greater therapeutic benefit in IgG-mediated inflammatory diseases compared with CTLA4-Ig alone.
Asunto(s)
Antígenos CD28/metabolismo , Antígeno CTLA-4/antagonistas & inhibidores , Inhibidores de Puntos de Control Inmunológico/farmacología , Proteína Coestimuladora de Linfocitos T Inducibles/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Abatacept/farmacología , Animales , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Femenino , Centro Germinal/efectos de los fármacos , Centro Germinal/metabolismo , Inmunidad Humoral/efectos de los fármacos , Inmunoglobulina G/metabolismo , Ligando Coestimulador de Linfocitos T Inducibles/metabolismo , Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Linfocitos T/metabolismoRESUMEN
α-Klotho, an aging-related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α-Klotho are a hallmark of age-associated diseases. Detecting or labeling α-Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by single-shot parallel automated fast-flow synthesis that recognize α-Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α-Klotho in physiological settings.
Asunto(s)
Glucuronidasa , Proteínas Klotho , Glucuronidasa/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Péptidos/metabolismo , Riñón/metabolismoRESUMEN
The kidney, parathyroid gland, and choroid plexus express the aging-related transmembrane protein α-Klotho, a coreceptor of the fibroblast growth factor 23 (FGF23) receptor complex. Reduced α-Klotho levels are correlated with chronic kidney disease and other age-related diseases, wherein they are released from membranes into circulation. Klotho's potential physiological action as a hormone is of current scientific interest. Part of the challenges associated with advancing these studies, however, has been the long-standing difficulty in detecting soluble α-Klotho in biofluids. Here, we describe the discovery of peptides that recognize α-Klotho with high affinity and selectivity by applying in-solution size-exclusion-based affinity selection-mass spectrometry (AS-MS). After two rounds of AS-MS and subsequent N-terminal modifications, the peptides improved their binding affinity to α-Klotho by approximately 2300-fold compared to the reported starting peptide Pep-10, previously designed based on the C-terminal region of FGF23. The lead peptide binders were shown to enrich α-Klotho from cell lysates and to label α-Klotho in kidney cells. Our results further support the utility of in-solution, label-free AS-MS protocols to discover peptide-based binders to target proteins of interest with high affinity and selectivity, resulting in functional probes for biological studies.
RESUMEN
BACKGROUND: Structure-based drug design (SBDD) can accelerate inhibitor lead design and optimization, and efficient methods including protein purification, characterization, crystallization, and high-resolution diffraction are all needed for rapid, iterative structure determination. Janus kinases are important targets that are amenable to structure-based drug design. Here we present the first mouse Tyk2 crystal structures, which are complexed to 3-aminoindazole compounds. RESULTS: A comprehensive construct design effort included N- and C-terminal variations, kinase-inactive mutations, and multiple species orthologs. High-throughput cloning and expression methods were coupled with an abbreviated purification protocol to optimize protein solubility and stability. In total, 50 Tyk2 constructs were generated. Many displayed poor expression, inadequate solubility, or incomplete affinity tag processing. One kinase-inactive murine Tyk2 construct, complexed with an ATP-competitive 3-aminoindazole inhibitor, provided crystals that diffracted to 2.5-2.6 Å resolution. This structure revealed initial "hot-spot" regions for SBDD, and provided a robust platform for ligand soaking experiments. Compared to previously reported human Tyk2 inhibitor crystal structures (Chrencik et al. (2010) J Mol Biol 400:413), our structures revealed a key difference in the glycine-rich loop conformation that is induced by the inhibitor. Ligand binding also conferred resistance to proteolytic degradation by thermolysin. As crystals could not be obtained with the unliganded enzyme, this enhanced stability is likely important for successful crystallization and inhibitor soaking methods. CONCLUSIONS: Practical criteria for construct performance and prioritization, the optimization of purification protocols to enhance protein yields and stability, and use of high-throughput construct exploration enable structure determination methods early in the drug discovery process. Additionally, specific ligands stabilize Tyk2 protein and may thereby enable crystallization.
Asunto(s)
Diseño de Fármacos , Indazoles/química , Indazoles/farmacología , TYK2 Quinasa/antagonistas & inhibidores , TYK2 Quinasa/química , Secuencia de Aminoácidos , Animales , Cristalización , Cristalografía por Rayos X , Estabilidad de Enzimas/efectos de los fármacos , Humanos , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/metabolismo , Ratones , Datos de Secuencia Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Estructura Secundaria de Proteína , Proteolisis/efectos de los fármacos , Relación Estructura-Actividad , TYK2 Quinasa/aislamiento & purificaciónRESUMEN
MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.
Asunto(s)
Antiinflamatorios/química , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/química , Adenosina Trifosfato/química , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacología , Sitios de Unión , Unión Competitiva , Simulación por Computador , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Pirimidinas/síntesis química , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
BACKGROUND: Structure-based drug design (SBDD) can provide valuable guidance to drug discovery programs. Robust construct design and expression, protein purification and characterization, protein crystallization, and high-resolution diffraction are all needed for rapid, iterative inhibitor design. We describe here robust methods to support SBDD on an oral anti-cytokine drug target, human MAPKAP kinase 2 (MK2). Our goal was to obtain useful diffraction data with a large number of chemically diverse lead compounds. Although MK2 structures and structural methods have been reported previously, reproducibility was low and improved methods were needed. RESULTS: Our construct design strategy had four tactics: N- and C-terminal variations; entropy-reducing surface mutations; activation loop deletions; and pseudoactivation mutations. Generic, high-throughput methods for cloning and expression were coupled with automated liquid dispensing for the rapid testing of crystallization conditions with minimal sample requirements. Initial results led to development of a novel, customized robotic crystallization screen that yielded MK2/inhibitor complex crystals under many conditions in seven crystal forms. In all, 44 MK2 constructs were generated, ~500 crystals were tested for diffraction, and ~30 structures were determined, delivering high-impact structural data to support our MK2 drug design effort. CONCLUSION: Key lessons included setting reasonable criteria for construct performance and prioritization, a willingness to design and use customized crystallization screens, and, crucially, initiation of high-throughput construct exploration very early in the drug discovery process.
Asunto(s)
Diseño de Fármacos , Péptidos y Proteínas de Señalización Intracelular/química , Mutagénesis Sitio-Dirigida , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Sustitución de Aminoácidos , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Conformación Proteica , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
BACKGROUND: CD40 is a 48 kDa type I transmembrane protein that is constitutively expressed on hematopoietic cells such as dendritic cells, macrophages, and B cells. Engagement of CD40 by CD40L expressed on T cells results in the production of proinflammatory cytokines, induces T helper cell function, and promotes macrophage activation. The involvement of CD40 in chronic immune activation has resulted in CD40 being proposed as a therapeutic target for a range of chronic inflammatory diseases. CD40 antagonists are currently being explored for the treatment of autoimmune diseases and several anti-CD40 agonist mAbs have entered clinical development for oncological indications. RESULTS: To better understand the mode of action of anti-CD40 mAbs, we have determined the x-ray crystal structures of the ABBV-323 (anti-CD40 antagonist, ravagalimab) Fab alone, ABBV-323 Fab complexed to human CD40 and FAB516 (anti-CD40 agonist) complexed to human CD40. These three crystals structures 1) identify the conformational CD40 epitope for ABBV-323 recognition 2) illustrate conformational changes which occur in the CDRs of ABBV-323 Fab upon CD40 binding and 3) develop a structural hypothesis for an agonist/antagonist switch in the LCDR1 of this proprietary class of CD40 antibodies. CONCLUSIONS: The structure of ABBV-323 Fab demonstrates a unique method for antagonism by stabilizing the proposed functional antiparallel dimer for CD40 receptor via novel contacts to LCDR1, namely residue position R32 which is further supported by a closely related agonist antibody FAB516 which shows only monomeric recognition and no contacts with LCDR1 due to a mutation to L32 on LCDR1. These data provide a structural basis for the full antagonist activity of ABBV-323.
Asunto(s)
Anticuerpos Monoclonales/química , Complejo Antígeno-Anticuerpo/química , Antígenos CD40/agonistas , Antígenos CD40/antagonistas & inhibidores , Antígenos CD40/química , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Modelos Moleculares , Transducción de Señal , Electricidad EstáticaRESUMEN
The enzyme 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase catalyses the first step in aromatic amino acid biosynthesis in prokaryotes, plants and fungi. Cells of Saccharomyces cerevisiae contain two catalytically redundant DAHP synthases, encoded by the genes ARO3 and ARO4, whose activities are feedback-inhibited by phenylalanine and tyrosine, respectively. ARO3/4 gene transcription is controlled by GCN4. The authors previously cloned an ARO3 gene orthologue from Candida albicans and found that: (1) it can complement an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess phenylalanine, and (2) a homozygous aro3-deletion mutant of C. albicans is phenotypically Aro(+), suggesting the existence of another isozyme(s). They now report the identification and functional characterization of the C. albicans orthologue of S. cerevisiae Aro4p. The two Aro4p enzymes share 68% amino acid identity. Phylogenetic analysis places the fungal DAHP synthases in a cluster separate from prokaryotic orthologues and suggests that ARO3 and ARO4 arose from a single gene via a gene duplication event early in fungal evolution. C. albicans ARO4 mRNA is elevated upon amino acid starvation, consistent with the presence of three putative Gcn4p-responsive elements (GCREs) in the gene promoter sequence. C. albicans ARO4 complements an aro3 aro4 double mutation in S. cerevisiae, an effect inhibited by excess tyrosine. The authors engineered Deltaaro3/Deltaaro3 Deltaaro4/MET3p::ARO4 cells of C. albicans (with one wild-type copy of ARO4 placed under control of the repressible MET3 promoter) and found that they fail to grow in the absence of aromatic amino acids when ARO4 expression is repressed, and that this growth defect can be partially rescued by aromatic amino acids and certain aromatic amino acid pathway intermediates. It is concluded that, like S. cerevisiae, C. albicans contains two DAHP synthases required for the first step in the aromatic amino acid biosynthetic pathway.