RESUMEN
Combinatorial molecular therapy in pancreatic ductal adenocarcinoma (PDAC) has yielded largely disappointing results in clinical testing to-date as a multitude of adaptive resistance mechanisms is making selection of patients via molecular markers that capture essential, intersecting signaling routes challenging. Here, we report the scaffolding protein connector enhancer of kinase suppressor of Ras 1 (CNKSR1) as mediator of resistance to MAPK (MEK) inhibition. MEK inhibition in CNKSR1high cancer cells induces translocation of CNKSR1 to the plasma membrane where the scaffolding protein interacts with and stabilizes the phosphorylated form of AKT. CNKSR1-mediated AKT activation following MEK inhibition was associated with increased cellular p-PRAS40 levels and reduced nuclear translocation and cellular levels of FoxO1, a negative regulator of AKT signaling. In clinical PDAC specimens, high cytoplasmatic CNKSR1 levels correlated with increased cellular phospho-AKT and mTOR levels. Pharmacological co-blockade of AKT and MEK ranked top in induced synergies with MEK inhibition in CNKSR1high pancreas cancer cells among other inhibitor combinations targeting known CNKSR1 signaling. In vivo, CNKSR1high pancreatic tumors treated with AKT and MEK inhibitors showed improved outcome in the combination arm compared with single-agent treatment, an effect not observed in CNKSR1low models.Our results identify CNKSR1 as regulator of adaptive resistance to MEK inhibition by promoting crosstalk to AKT signaling via a scaffolding function for the phosphorylated form of AKT. CNSKR1 expression might be a possible molecular marker to enrich patients for future AKT-MEK inhibitor precision medicine studies. IMPLICATIONS: The CNKSR1 scaffold, identified within an RNAi screen as a novel mediator of resistance to MEK inhibition in pancreas cancer, connects the MAPK pathway and AKT signaling and may be adopted as a biomarker to select patients for combined MEK AKT blockade.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos , Línea Celular Tumoral , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias PancreáticasRESUMEN
Peptides were designed to inhibit the protein-protein interaction of CD2 and CD58 to modulate the immune response. This work involved the design and synthesis of eight different peptides by replacing each amino acid residue in peptide 6 with alanine as well as grafting the peptide to the sunflower trypsin-inhibitor framework. From the alanine scanning studies, mutation at position 2 of the peptide was shown to result in increased potency to inhibit cell adhesion interactions. The most potent peptide from the alanine scanning was further studied for its detailed three-dimensional structure and binding to CD58 protein using surface plasmon resonance and flow cytometry. This peptide was used to graft to the sunflower trypsin inhibitor to improve the stability of the peptide. The grafted peptide, SFTI-a1, was further studied for its potency as well as its thermal, chemical, and enzymatic stability. The grafted peptide exhibited improved activity compared to our previously grafted peptide and was stable against thermal and enzymatic degradation.
Asunto(s)
Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Sitios de Unión , Unión Competitiva , Antígenos CD2/química , Antígenos CD58/química , Adhesión Celular/efectos de los fármacos , Línea Celular , Diseño de Fármacos , Humanos , Simulación del Acoplamiento Molecular , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Unión Proteica , Mapas de Interacción de Proteínas/efectos de los fármacos , Estabilidad Proteica , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Inhibidores de Tripsina/farmacologíaRESUMEN
Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206high patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.
Asunto(s)
Lectinas de Unión a Manosa , Macrófagos Asociados a Tumores , Animales , Línea Celular Tumoral , Humanos , Inmunidad Innata , Lectinas Tipo C , Receptor de Manosa , Ratones , Receptores de Superficie CelularRESUMEN
Protein-protein interactions (PPI) by homo-, hetero- or oligo-merization in the cellular environment regulate cellular processes. PPI can be inhibited by antibodies, small molecules or peptides, and this inhibition has therapeutic value. A recently developed method, the proximity ligation assay (PLA), provides detection of PPI in the cellular environment. However, most applications using this assay are for proteins expressed in the same cell. We employ PLA for the first time to study PPI of cell surface proteins on two different cells. Inhibition of PPI using a peptide inhibitor is also quantified using this assay; PLA is used to detect PPI of CD2 and CD58 between Jurkat cells (T cells) and human fibroblast-like synoviocyte-rheumatoid arthritis cells that are important in the immune response in the autoimmune disease rheumatoid arthritis. This assay provides direct evidence of inhibition of PPI of two proteins on different cell surfaces.
Asunto(s)
Biotecnología/métodos , Antígenos de Histocompatibilidad Clase II/análisis , Proteínas de la Membrana/química , Proteínas/química , Antígenos CD2/análisis , Antígenos CD2/metabolismo , Antígenos CD58/análisis , Antígenos CD58/metabolismo , Células Cultivadas , Citometría de Flujo , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Células Jurkat , Proteínas de la Membrana/análisis , Modelos Moleculares , Unión Proteica , SinoviocitosRESUMEN
HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2-positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2-mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18) exhibited antiproliferative activity in HER2-overexpressing lung cancer cell lines with IC50 values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d-amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18, compound 32, a cyclic, d-amino acid-containing peptidomimetic, was found to have an IC50 value in the nanomolar range in HER2-overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d-amino acid analogs of 18, compound 32, binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.
Asunto(s)
Aminoácidos Cíclicos/farmacología , Antineoplásicos/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Peptidomiméticos/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-3/antagonistas & inhibidores , Secuencia de Aminoácidos , Aminoácidos Cíclicos/síntesis química , Antineoplásicos/síntesis química , Sitios de Unión , Unión Competitiva , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Expresión Génica , Humanos , Concentración 50 Inhibidora , Células MCF-7 , Peptidomiméticos/síntesis química , Unión Proteica , Estabilidad Proteica , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Colorectal cancer (CRC) is the third most common solid internal malignancy among cancers. Early detection of cancer is key to increasing the survival rate of colorectal cancer patients. Overexpression of the EGFR protein is associated with CRC. We have designed a series of peptides that are highly specific for the extracellular domain of EGFR, based on our earlier studies on linear peptides. The previously reported linear peptide LARLLT, known to bind to EGFR, was modified with the goals of increasing its stability and its specificity toward EGFR. Peptide modifications, including D-amino acid substitution, cyclization, and chain reversal, were investigated. In addition, to facilitate labeling of the peptide with a fluorescent dye, an additional lysine residue was introduced onto the linear (KLARLLT) and cyclic peptides cyclo(KLARLLT) (Cyclo.L1). The lysine residue was also converted into an azide group in both a linear and reversed cyclic peptide sequences cyclo(K(N3)larllt) (Cyclo.L1.1) to allow for subsequent "click" conjugation. The cyclic peptides showed enhanced binding to EGFR by SPR. NMR and molecular modeling studies suggest that the peptides acquire a ß-turn structure in solution. In vitro stability studies in human serum show that the cyclic peptide is more stable than the linear peptide.
Asunto(s)
Receptores ErbB/metabolismo , Oligopéptidos/metabolismo , Péptidos Cíclicos/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Estabilidad de Medicamentos , Receptores ErbB/química , Humanos , Enlace de Hidrógeno , Ligandos , Simulación del Acoplamiento Molecular , Oligopéptidos/química , Oligopéptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
Among different types of EGFR dimers, EGFR-HER2 and HER2-HER3 are well known in different types of cancers. Targeting dimerization of EGFR will have a significant impact on cancer therapies. A symmetric peptidomimetic was designed to inhibit the protein-protein interaction of EGFR. The peptidomimetic (Cyclo(1,10)PpR (R) Anapa-FDDF-(R)-Anapa)R, compound 18) was shown to exhibit antiproliferative activity with an IC50 of 194 nM in HER2-expressing breast cancer cell lines and 18 nM in lung cancer cell lines. The peptidomimetic has a Pro-Pro sequence in the structure to stabilize the ß-turn and a ß-amino acid, amino napthyl propionic acid. To investigate the effect of the chirality of ß-amino acid on the structure of the peptide and its antiproliferative activity, diastereoisomers of compound 18 were designed and synthesized. Structure-activity relationships of these compounds indicated that there is a chiral switch at ß-amino acid in the designed compound. The peptidomimetic with R configuration at ß-amino acid and with a L-Pro-D-Pro sequence was the most active compound (18). Using enzyme complement fragmentation assay and proximity ligation assay, we show that compound 18 inhibits HER2:HER3 and EGFR:HER2 dimerization. Surface plasmon resonance studies suggested that compound 18 binds to the HER2 extracellular domain and in particular to domain IV. The anticancer activity of compound 18 was evaluated using a xenograft model of breast cancer in mice; compound 18 suppressed the tumor growth in mice compared to control. Compound 18 was also shown to have a synergistic effect with erlotinib on EGFR mutated lung cancer cell lines.
RESUMEN
Nature provides a variety of peptides that are expressed in most living species. Evolutionary pressure and natural selection have created and optimized these peptides to bind to receptors with high affinity. Hence, natural resources provide an abundant chemical space to be explored in peptide-based drug discovery. Marine peptides can be extracted by simple solvent extraction techniques. The advancement of analytical techniques has made it possible to obtain pure peptides from natural resources. Extracted peptides have been evaluated as possible therapeutic agents for a wide range of diseases, including antibacterial, antifungal, antidiabetic and anticancer activity as well as cardiovascular and neurotoxin activity. Although marine resources provide thousands of possible peptides, only a few peptides derived from marine sources have reached the pharmaceutical market. This review focuses on some of the peptides derived from marine sources in the past ten years and gives a brief review of those that are currently in clinical trials or on the market.
Asunto(s)
Productos Biológicos/química , Productos Biológicos/farmacología , Péptidos/química , Péptidos/farmacología , Peptidomiméticos/química , Peptidomiméticos/farmacología , Animales , Organismos Acuáticos/química , Descubrimiento de Drogas/métodos , HumanosRESUMEN
Doxorubicin (DOX) belongs to the anthracycline class of drugs that are used in the treatment of various cancers. It has limited cystostatic effects in therapeutic doses, but higher doses can cause cardiotoxicity. In the current approach, we conjugated a peptidomimetic (Arg-aminonaphthylpropionic acid-Phe, compound 5) known to bind to HER2 protein to DOX via a glutaric anhydride linker. Antiproliferative assays suggest that the DOX-peptidomimetic conjugate has activity in the lower micromolar range. The conjugate exhibited higher toxicity in HER2-overexpressed cells than in MCF-7 and MCF-10A cells that do not overexpress HER2 protein. Cellular uptake studies using confocal microscope experiments showed that the conjugate binds to HER2-overexpressed cells and DOX is taken up into the cells in 4 h compared to conjugate in MCF-7 cells. Binding studies using surface plasmon resonance indicated that the conjugate binds to the HER2 extracellular domain with high affinity compared to compound 5 or DOX alone. The conjugate was stable in the presence of cells with a half-life of nearly 4 h and 1 h in human serum. DOX is released from the conjugate and internalized into the cells in 4 h, causing cellular toxicity. These results suggest that this conjugate can be used to target DOX to HER2-overexpressing cells and can improve the therapeutic index of DOX for HER2-positive cancer.
Asunto(s)
Antineoplásicos/farmacocinética , Doxorrubicina/farmacología , Peptidomiméticos/farmacología , Receptor ErbB-2/análisis , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Doxorrubicina/química , Doxorrubicina/farmacocinética , Femenino , Semivida , Humanos , Células MCF-7 , Peptidomiméticos/química , Peptidomiméticos/farmacocinética , Unión ProteicaRESUMEN
The interaction between the cell-cell adhesion proteins CD2 and CD58 plays a crucial role in lymphocyte recruitment to inflammatory sites, and inhibitors of this interaction have potential as immunomodulatory drugs in autoimmune diseases. Peptides from the CD2 adhesion domain were designed to inhibit CD2:CD58 interactions. To improve the stability of the peptides, ß-sheet epitopes from the CD2 region implicated in CD58 recognition were grafted into the cyclic peptide frameworks of sunflower trypsin inhibitor and rhesus theta defensin. The designed multicyclic peptides were evaluated for their ability to modulate cell-cell interactions in three different cell adhesion assays, with one candidate, SFTI-a, showing potent activity in the nanomolar range (IC50: 51 nM). This peptide also suppresses the immune responses in T cells obtained from mice that exhibit the autoimmune disease rheumatoid arthritis. SFTI-a was resistant to thermal denaturation, as judged by circular dichroism spectroscopy and mass spectrometry, and had a half-life of â¼24 h in human serum. Binding of this peptide to CD58 was predicted by molecular docking studies and experimentally confirmed by surface plasmon resonance experiments. Our results suggest that cyclic peptides from natural sources are promising scaffolds for modulating protein-protein interactions that are typically difficult to target with small-molecule compounds.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Péptidos Cíclicos/farmacología , Secuencia de Aminoácidos , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Dicroismo Circular , Humanos , Espectrometría de Masas , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Péptidos Cíclicos/química , Unión Proteica , Resonancia por Plasmón de SuperficieRESUMEN
CD2 and CD58 are two important costimulatory molecules involved in generating the signal II required for normal immune signaling. However, this interaction can be targeted to be of benefit in cases of abnormal immune signaling seen in autoimmune diseases. Our objective in this study was to design a peptidomimetic (compound 7) based on a ß-strand structure of the adhesion domain of CD2 protein to inhibit CD2-CD58 protein-protein interaction and its effect on immunomodulation in the collagen-induced arthritis (CIA) model. The ability of compound 7 to bind to CD58 protein was assessed using flow cytometry. The effect of compound 7 on modulating the immune response was evaluated in an autoimmune disease using CIA in mice. The stability of compound 7 was evaluated in mouse serum using mass spectrometry. Antibody (Ab) binding inhibition studies suggested that compound 7 binds to CD58 protein. Compound 7 was successful in modulating immune responses when administered in the CIA mouse model along with reducing anti-collagen Ab levels and decreasing the level of interferon gamma (IFN-γ) relative to control treatments. Compound 7 was found to be nonimmunogenic and stable in mouse serum up to 48 h. Results suggest that compound 7 can serve as a lead compound for immunomodulation, and could be a therapeutic agent for the autoimmune disease rheumatoid arthritis (RA).
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Antígenos CD2/inmunología , Antígenos CD58/inmunología , Adhesión Celular/efectos de los fármacos , Colágeno/efectos de los fármacos , Peptidomiméticos/uso terapéutico , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Unión Competitiva , Colágeno/inmunología , Femenino , Ratones , Ratones Endogámicos DBA , Peptidomiméticos/farmacologíaRESUMEN
Blocking protein-protein interactions (PPI) using small molecules or peptides modulates biochemical pathways and has therapeutic significance. PPI inhibition for designing drug-like molecules is a new area that has been explored extensively during the last decade. Considering the number of available PPI inhibitor databases and the limited number of 3D structures available for proteins, docking and scoring methods play a major role in designing PPI inhibitors as well as stabilizers. Docking methods are used in the design of PPI inhibitors at several stages of finding a lead compound, including modeling the protein complex, screening for hot spots on the protein-protein interaction interface and screening small molecules or peptides that bind to the PPI interface. There are three major challenges to the use of docking on the relatively flat surfaces of PPI. In this review we will provide some examples of the use of docking in PPI inhibitor design as well as its limitations. The combination of experimental and docking methods with improved scoring function has thus far resulted in few success stories of PPI inhibitors for therapeutic purposes. Docking algorithms used for PPI are in the early stages, however, and as more data are available docking will become a highly promising area in the design of PPI inhibitors or stabilizers.