Seguridad y eficacia del uso consecutivo de afatinib y erlotinib en el tratamiento de primera línea de cáncer de pulmón de células no pequeñas (nsclc) en pacientes con mutacion en el gen del receptor del factor de crecimiento epidérmico (EFGR), que hayan respondido previamente al tratamiento con uno de ellos / Safety and efficacy of consecutive use of afatinib and erlotinib in the first-line treatment of non-small cell lung cancer (nsclc) in patients with mutation in the epidermal growth factor receptor gene (egfr) who have previously responded to treatmen

En el Dictamen Preliminar de Evaluación de Tecnología Sanitaria N° 002-SDEPFyOTS-DETS IETSI-2015, luego de concluir que no existían diferencias en relación a la eficacia y seguridad entre afatinib y erlotinib, se decide aprobar el uso de erlotinib por tener un menor costo en el mercado. A partir de la aprobación del uso de erlotinib, surge una nueva pregunta a resolver con respecto a los pacientes que ya se encontraban en tratamiento con afatinib extra institucionalmente. De hecho, existen situaciones como ésta en las que se plantea el cambio de un TKI a otro, aún en condiciones en las que se ha logrado buena evolución clínica y en ausencia de efectos adversos serios al TKI de inicio, por razones más bien extramédicas (como también pueden ser cambios en la disponibilidad del TKI y costos). Así, se plantea la pregunta si el intercambio entre TKIs en dichas condiciones mantiene la respuesta lograda con el primer TKI. \tEs por ello que el presente dictamen expone la evaluación de la eficacia y seguridad del uso consecutivo, de dos TKIs distintos (i.e afatinib y erlotinib) para el tratamiento de primera línea de NSCLC avanzando (estadio 111E3) o metastásico (estadio IV) en pacientes con mutaciones en el gen EGFR, que hayan obtenido respuesta completa sin toxicidad inaceptable previamente al tratamiento con un TKI. En la presente evaluación sobre el uso consecutivo de dos TKIs (i.e afatinib y erlotinib) no se ha encontrado evidencia directa que evalúe la eficacia y seguridad sostenida al intercambiar TKIs para el tratamiento de primera línea de NSCLC en pacientes con mutación en EGFR, en pacientes que han respondido a uno de dichos TKIs sin presentar toxicidad inaceptable. -\tTomando en consideración la evidencia expuesta en el dictamen N° 002-SDEPFyOTS-DETS IETSI-2015 donde no se encontraron diferencias entre afatinib y erlotinib a nivel clínico con respecto a su eficacia y seguridad, es razonable esperar por analogía que ambos fármacos al ser similares pueden ser intercambiables con un bajo riesgo de que se modifique el curso clínico de la enfermedad logrado con el TKI inicial. La población de interés en la presente evaluación de tecnología son los pacientes que han presentado una respuesta excepcional al TKI utilizado (i.e., respuesta completa sin toxicidad importante). En estos casos, y considerando la ausencia de evidencia directa que demuestre que el intercambio de TKIs, en el contexto de no progresión, mantiene la seguridad y eficacia del TKI ya iniciado, el grado de incertidumbre escala a un mayor nivel de complejidad. Por lo tanto, se acude a la opinión de expertos. Así, en opinión de expertos se establece que ante la ausencia de evidencia directa que respalde el intercambio de TKIs y en los casos de pacientes que presentan una respuesta completa al tratamiento, lo mejor es intentar mantener la terapia con la que se ha logrado dicha respuesta y no correr el riesgo, aunque éste sea bajo, de que al cambiar de tratamiento de un TKI a otro, el curso de esta respuesta excepcional varíe. Por lo expuesto, el Instituto de Evaluación de Tecnologías en Salud e Investigación ­ IETSI, aprueba por el periodo de un año a partir de la fecha de publicación del presente Dictamen Preliminar, el uso de afatinib en pacientes con cáncer de pulmón de células no pequeñas (NSCLC) con mutación del gen EGFR, con enfermedad localmente avanzada o metastásica, que presentan respuesta completa sin toxicidad inaceptable con afatinib recibido extra institucionalmente (Anexo 01). Notar que todos aquellos pacientes nuevos con diagnóstico de NSCLC con mutación en el gen EGFR, y aquellos que no cumplen los criterios de respuesta completa al afatinib sin toxicidad inaceptable, deberán acogerse al Dictamen Preliminar N° 002-SDEPFyOTS-DETS IETSI-2015. Los Comités Farmacoterapéuticos de las redes asistenciales deberán evaluar estos casos de manera exhaustiva, bajo responsabilidad, teniendo en cuenta que el presente Dictamen Preliminar no reemplaza al Dictamen Preliminar N° 002-SDEPFyOTS-DETS IETSI-2015, dado que cada dictamen responde a una población diferente. Dado que la evidencia que respalda el intercambio de TKIs, es aún limitada, se establece que el efecto de dicho intercambio se evaluará con los datos de los pacientes que hayan recibido afatinib, bajo las condiciones del presente dictamen. Esta información será tomada en cuenta en la reevaluación de este medicamento al terminar la vigencia de este Dictamen Preliminar (un año a partir de la fecha de publicación).(AU)

Development of an epidermal growth factor derivative with EGFR blocking activity.

The members of the epidermal growth factor (EGF)/ErbB family are prime targets for cancer therapy. However, the therapeutic efficiency of the existing anti-ErbB agents is limited. Thus, identifying new molecules that inactivate the ErbB receptors through novel strategies is an important goal on cancer research. In this study we have developed a shorter form of human EGF (EGFt) with a truncated C-terminal as a novel EGFR inhibitor. EGFt was designed based on the superimposition of the three-dimensional structures of EGF and the Potato Carboxypeptidase Inhibitor (PCI), an EGFR blocker previously described by our group. The peptide was produced in E. coli with a high yield of the correctly folded peptide. EGFt showed specificity and high affinity for EGFR but induced poor EGFR homodimerization and phosphorylation. Interestingly, EGFt promoted EGFR internalization and translocation to the cell nucleus although it did not stimulate the cell growth. In addition, EGFt competed with EGFR native ligands, inhibiting the proliferation of cancer cells. These data indicate that EGFt may be a potential EGFR blocker for cancer therapy. In addition, the lack of EGFR-mediated growth-stimulatory activity makes EGFt an excellent delivery agent to target toxins to tumours over-expressing EGFR.

Dual cleavage of neuregulin 1 type III by BACE1 and ADAM17 liberates its EGF-like domain and allows paracrine signaling.

Proteolytic shedding of cell surface proteins generates paracrine signals involved in numerous signaling pathways. Neuregulin 1 (NRG1) type III is involved in myelination of the peripheral nervous system, for which it requires proteolytic activation by proteases of the ADAM family and BACE1. These proteases are major therapeutic targets for the prevention of Alzheimer's disease because they are also involved in the proteolytic generation of the neurotoxic amyloid ß-peptide. Identification and functional investigation of their physiological substrates is therefore of greatest importance in preventing unwanted side effects. Here we investigated proteolytic processing of NRG1 type III and demonstrate that the ectodomain can be cleaved by three different sheddases, namely ADAM10, ADAM17, and BACE1. Surprisingly, we not only found cleavage by ADAM10, ADAM17, and BACE1 C-terminal to the epidermal growth factor (EGF)-like domain, which is believed to play a pivotal role in signaling, but also additional cleavage sites for ADAM17 and BACE1 N-terminal to that domain. Proteolytic processing at N- and C-terminal sites of the EGF-like domain results in the secretion of this domain from NRG1 type III. The soluble EGF-like domain is functionally active and stimulates ErbB3 signaling in tissue culture assays. Moreover, the soluble EGF-like domain is capable of rescuing hypomyelination in a zebrafish mutant lacking BACE1. Our data suggest that NRG1 type III-dependent myelination is not only controlled by membrane-retained NRG1 type III, but also in a paracrine manner via proteolytic liberation of the EGF-like domain.

6-alkynyl fucose is a bioorthogonal analog for O-fucosylation of epidermal growth factor-like repeats and thrombospondin type-1 repeats by protein O-fucosyltransferases 1 and 2.

Protein O-fucosyltransferase 1 (Pofut1) and protein O-fucosyltransferase 2 (Pofut2) add O-linked fucose at distinct consensus sequences in properly folded epidermal growth factor (EGF)-like repeats and thrombospondin type-1 (TSR) repeats, respectively. Glycan chain elongation past O-fucose can occur to yield a tetrasaccharide on EGF repeats and a disaccharide on TSRs. Elimination of Pofut1 in mice causes embryonic lethality with Notch-like phenotypes demonstrating that O-fucosylation of Notch is essential for its function. Similarly, elimination of Pofut2 results in an early embryonic lethal phenotype in mice, although the molecular mechanism for the lethality is unknown. The recent development of sugar analogs has revolutionized the study of glycans by providing a convenient method for labeling and tracking glycosylation. In order to study O-fucosylation, we took advantage of the recently developed reporter, 6-alkynyl fucose. Using the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), or "click" reaction, azido-biotin allows tagging and detection of 6AF-modified proteins. Here we examine whether proteins containing EGF repeats or TSRs with O-fucose consensus sequences are specifically modified with 6AF in cell culture. Using mass spectrometry (MS), we demonstrate that 6AF is efficiently incorporated onto the appropriate consensus sequences on EGF repeats and TSRs. Furthermore, the elongation of the O-fucose monosaccharide on EGF repeats and TSRs is not hampered when 6AF is used. These results show that 6AF is efficiently utilized in a truly bioorthogonal manner by Pofut1, Pofut2 and the enzymes that elongate O-fucose, providing evidence that 6AF is a significant new tool in the study of protein O-fucosylation.

The release of EGF domain from EGF-like factors by a specific cleavage enzyme activates the EGFR-MAPK3/1 pathway in both granulosa cells and cumulus cells during the ovulation process.

In mammalian preovulatory follicles, LH stimulation induces the ovulation process, including follicular wall rupture, granulosa cell luteinization, cumulus cell expansion and meiotic maturation of the oocyte. The receptor for LH (LHCGR) is expressed mostly in granulosa cells of preovulatory follicles, and is rarely expressed in cumulus cells or oocytes. The expression level in granulosa cells dramatically decreases after ovulation stimuli. Thus, a potent factor(s) secreted by granulosa cells is required to stimulate not only granulosa cells via an autocrine manner but also cumulus cells and/or oocytes via a paracrine pathway. Recent reports showed that granulosa cells and cumulus cells express EGF-like factors that activate the EGF receptor (EGFR)-mitogen-activated protein kinase3/1 (MAPK3/1) (also known as extracellular signal-regulated kinase1/2 (ERK1/2)) pathway in both cell types. EGF-like factors are composed of a signal sequence, transmembrane domain and EGF domain, suggesting that release of the EGF domain by a specific enzyme is essential for interaction with the EGFR to induce the ovulation process. In our studies, TACE/ADAM17, which is known to be a proteolytic enzyme of EGF-like factors in many types of tissue, was found to be expressed in FSH/LH-stimulated granulosa cells and cumulus cells together with activation of the EGFR-MAPK3/1 pathway. When TACE/ADAM17 activity was decreased by a specific inhibitor or siRNA technique, granulosa cell luteinization, cumulus expansion and oocyte maturation were suppressed in an in vitro culture. Thus, TACE/ADAM17 is one of the key genes expressed in both granulosa cells and cumulus cells for induction of the ovulation process.

Dendrimer-epidermal growth factor conjugate displays superagonist activity.

Binding of ligands on to epidermal growth factor receptor (EGFR) can stimulate cell growth; therefore, any application employing EGF as a targeting ligand for a "drug carrier" must evaluate the effect of the conjugate on cell growth. We report the synthesis and in vitro biological activity of EGF molecules coupled to a fluorescein-labeled polyamidoamine dendrimer. The conjugate bound and internalized into several EGFR-expressing cell lines in a receptor-specific fashion. The conjugate effectively induced EGFR phosphorylation and acted as a superagonist by stimulating cell growth to a greater degree than free EGF. Concomitant administration of the chemotherapeutic drug methotrexate completely inhibited cell growth to a degree similar to its effect in the absence of the conjugate. Thus, dendrimer-EGF conjugates serve as EGFR superagonists, but this activity can be overcome by chemotherapeutic drugs. The agonist activity of these materials must be taken into consideration when using EGF conjugates for imaging applications.

In vivo administration of epidermal growth factor and its homologue attenuates developmental maturation of functional excitatory synapses in cortical GABAergic neurons.

The ErbB1 ligand family includes epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha), heparin-binding EGF-like growth factor, amphiregulin and betacellulin. Previously, we demonstrated that TGFalpha decreases alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors in cultured neocortical gamma-aminobutyric acid (GABA) neurons. In the present study, we examined in vivo effects of EGF and TGFalpha in the mouse neocortex using electrophysiological and biochemical techniques. In mouse neonates, subcutaneously administered EGF penetrated the blood-brain barrier and activated ErbB1 in the neocortex. Daily administration of EGF or TGFalpha attenuates developmental increases in expression of the AMPA receptor subunits (GluR1 and GluR2/3) in the neocortex of postnatal mice. Immunohistochemistry revealed that the reduction in AMPA receptor expression was significant in the GABAergic neurons, especially those positive for parvalbumin. Using cortical slices prepared from EGF-treated mice, we recorded miniature excitatory postsynaptic currents (mEPSCs) in both GABAergic and pyramidal neurons. Subchronic treatment with EGF decreased the amplitude and frequency of mEPSCs in GABAergic neurons, but its effects were negligible on pyramidal neurons. We conclude that EGF or other ErbB1 ligand(s) attenuates a developmental increase in AMPA receptor expression and function in cortical GABAergic neurons.

In vivo detection of head and neck cancer orthotopic xenografts by immunofluorescence.

PURPOSE: To determine whether Cy5.5-labeled antiepidermal growth factor (EGFR) antibody could be used to detect head and neck squamous cell carcinoma (HNSCC) xenografts in vivo. METHODS: AntiEGFR antibody (cetuximab) was labeled with Cy5.5, a fluorophore with emission in the near infrared range. The cetuximab-Cy5.5 conjugate was systemically administered in subtherapeutic doses (50 microg) to mice bearing orthotopically xenografted HNSCC cell lines (SCC1, CAL27, and FaDu). As a control, isotype-matched human immunoglobulin (Ig)G1k antibody labeled with Cy5.5 was systemically injected in parallel experiments. All tumor regions (n = 6) were imaged by fluorescent stereomicroscopy at 0, 6, 24, 48, or 72 hours. Tumor size was measured by high-frequency ultrasonography at 72 hours. Transcervical partial and near-total resections were then performed with stereomicroscopic imaging after each resection. The mandible and associated structures were then resected, paraffin embedded, and then serial sectioned for analysis. RESULTS: Tumors could be clearly visualized by near infrared fluorescent stereomicroscopy at 48 and 72 hours after systemic administration of cetuximab-Cy5.5 but not after administration with the labeled isotype control antibody, IgG1k-Cy5.5. Ultrasound measurement of tumors (n = 5) correlated with fluorescent measurements of tumor (Spearman's coefficient, 0.92, P

Preparation and evaluation of (68)Ga-DOTA-hEGF for visualization of EGFR expression in malignant tumors.

UNLABELLED: Detection of epidermal growth factor receptor (EGFR) overexpression in many carcinomas provides important diagnostic information, which can influence patient management. The use of PET may enable such detection in vivo by a noninvasive procedure with high sensitivity. The aim of this study was to develop a method for preparation of a positron-emitting tracer based on a natural ligand to EGFR, the recombinant human epidermal growth factor (hEGF), and to perform a preclinical evaluation of the tracer. METHODS: DOTA-hEGF (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was prepared by coupling of a N-sulfosuccinimide ester of DOTA to hEGF. The conjugate was labeled with a generator-produced positron-emitting nuclide, (68)Ga (half-life = 68 min), using microwave heating. Binding specificity, affinity, internalization, and retention of (68)Ga-DOTA-hEGF was studied in 2 EGFR-expressing cell lines, U343 glioma cells and A431 cervical carcinoma cells. Biodistribution and microPET visualization studies were performed in BALB/c nu/nu mice bearing A431 carcinoma xenografts. RESULTS: A 1-min-long microwave-assisted labeling provided radioactivity incorporation of 77% +/- 4%. Both cell lines demonstrated receptor-specific uptake of the conjugate, rapid internalization of the tracer, and good retention of radioactivity. Binding to both cell lines occurred with high affinity, approximately 2 nmol/L. The biodistribution study demonstrated accumulation of radioactivity in xenografts and in EGFR-expressing organs. The microPET imaging study enabled visualization of tumors and demonstrated quick--within 5 min--localization of radioactivity in tumors. CONCLUSION: (68)Ga-DOTA-hEGF has potential for imaging EGFR overexpression in tumors.

Near-infrared optical imaging of epidermal growth factor receptor in breast cancer xenografts.

The specificity of a novel epidermal growth factor (EGF)-Cy5.5 fluorescent optical probe in the detection of EGF receptor (EGFr) was assessed using continuous-wave fluorescence imaging accomplished via an intensified charge-coupled device (CCD) camera. Human mammary MDA-MB-468 (EGFr+) and MDA-MB-435 (EGFr-) cancer cells were incubated with Cy5.5, EGF-Cy5.5, or the anti-EGFr monoclonal antibody C225 or EGF followed by EGF-Cy5.5 and examined under a fluorescence microscope. In vivo imaging was performed on mice with s.c. MDA-MB-468 and MDA-MB-435 tumors. Images were obtained every 6 s for 20 min after i.v. injection of each agent and every 24 h after injection for up to 192 h. Additionally, mice with MDA-MB-468 tumors were injected i.v. with C225 24 h before injection of EGF-Cy5.5. EGF-Cy5.5, but not Cy5.5 or indocyanine green dye (ICG), bound to MDA-MB-468 cells. Binding of EGF-Cy5.5 was blocked by C225 and by EGF. In contrast, binding of EGF-Cy5.5 to MDA-MB-435 cells was not observed. Monitoring of the time-fluorescence intensity in mice confirmed that ICG and Cy5.5 had no favorable binding to tumor regardless of EGFr expression level. In contrast, EGF-Cy5.5 accumulated only in MDA-MB-468 tumors. Moreover, tumor uptake of EGF-Cy5.5 was blocked by C225. ICG and Cy5.5 fluorescence was completely absent from the tumor site, regardless of EGFr expression level, 24 h after injection. Little EGF-Cy5.5 fluorescence was detected in MDA-MB-435 tumors 24 h after injection. In MDA-MB-468 tumors, our data suggest that EGF-Cy5.5 may be used as a specific NIR contrast agent for noninvasive imaging of EGFr expression and monitoring of responses to molecularly targeted therapy.

[(111)In]Bz-DTPA-hEGF: Preparation and in vitro characterization of a potential anti-glioblastoma targeting agent.

The overexpression of epidermal growth factor receptors, EGFR, in glioblastomas is well documented. Hence, the EGFR can be used as target structure for a specific targeting of glioblastomas. Both radiolabeled anti-EGFR antibodies and the natural ligand EGF are candidate agents for targeting. However, EGF, which has a rather low molecular weight (6 kDa), might have better tissue penetration properties through both normal tissue and tumors in comparison with anti-EGF antibodies and their fragments. The aim of this study was to prepare and evaluate in vitro an EGF-based antiglioma conjugate with residualizing label. Human recombinant EGF (hEGF) was coupled to isothiocyanate-benzyl-DTPA. The conjugate was purified from unreacted chelator using solid-phase extraction and labeled with (111)In. The labeling yield was 87% +/- 7%. The label was reasonably stable; the transchelation of (111)In to serum proteins was about 5% after incubation at 37 degrees C during 24 hours. The obtained [(111)In]benzyl-DTPA-hEGF conjugate was characterized in vitro using the EGFR expressing glioma cell line U343MGaCl2:6. The binding affinity, internalization, and retention of the conjugate were studied. The conjugate had receptor specific binding and the radioactivity was quickly internalized. The intracellular retention of radioactivity after interrupted incubation with conjugate was 71% +/- 1% and 59% +/- 1.5% at 24 and 45 hours, respectively. The dissociation constant was estimated to 2.0 nM. The results indicate that [(111)In]benzyl-DTPA-hEGF is a potential candidate for targeting glioblastoma cells, possibly using locoregional injection.

Rapid microscale enzymic semisynthesis of epidermal growth factor (EGF) analogues.

Epidermal Growth Factor (EGF) is a small growth factor containing 53 amino acid residues capable of stimulating the proliferation of both mesenchymal and epithelial cells. Comparison of the amino acid sequences of EGF from several species, and related proteins that can bind to the EGF receptor (e.g. TGFalpha, VGF, heparin-binding EGF, and betacellulin), suggests that Leu47, which is highly conserved, is important for biological function. Additionally, we have shown previously, using a combination of trypsin and carboxypeptidase Y digestion of native murine EGF, that removal of Leu47 results in more than 100-fold decrease in both receptor binding and mitogenic activity. We now describe a micromethod for the rapid generation of C-terminally modified EGFs to investigate further the role of C-terminal residues in determining functional activity. These analogues have been generated by digesting native murine EGF with trypsin, purifying the biologically inactive, but structurally intact, EGF1-45 core by micropreparative RP-HPLC, and then reversing the action of trypsin to couple synthetic peptides (e.g. DL, DI, DF, EL, DLLW) onto the C-terminus of the EGF1-45 core. This enzymic semisynthesis method allows multiple derivatives to be generated rapidly from microgram quantities of EGF1-45 in sufficient quantities for sensitive biological and physicochemical analysis. We have validated the method by regenerating EGF1-47 from EGF1-45 with equivalent mitogenic and receptor binding activity to EGF1-47 generated from wild type EGF by digestion with trypsin and carboxypeptidase Y. We have also investigated the effect of substituting alternative normal or nonphysiological amino acids (e.g. allo-Ile) for Asp46, Leu47 or Arg48. Even small changes in these C-terminal residues reduce the mitogenic potency of the analogue.

Antitumor activity of alpha fetoprotein and epidermal growth factor conjugates in vitro and in vivo.

Conjugates of carminomycin (Cm) with alpha-fetoprotein (AFP) and epidermal growth factor (EGF) were prepared and their cytotoxic activities were studied in vitro. Both conjugates showed cytotoxic activity which exceeded that of free Cm in tumor cell cultures of MCF-7, SKOV3, QOS, P388 and B16 cells. The antitumor effects of the conjugates were studied in vivo in mice with subcutaneous tumors of B16 and P388 cells. The Cm-AFP and Cm-EGF conjugates inhibited tumor growth and noticeably increased the mean life span in experimental animals. Our results suggest that the therapeutic activity of Cm can be significantly enhanced by conjugation to AFP or EGF.

The phosphatase domains of LAR, CD45, and PTP1B: structural correlations with peptide-based inhibitors.

PTP1B is a cytosolic protein tyrosine phosphatase that is a regulator of the kinase activity of the insulin receptor; the two protein tyrosine phosphatases LAR and CD45 are receptor type phosphatases crucially important to cell function. LAR also is involved in regulation of the insulin receptor while CD45 is critical for T-cell activation. Although LAR and CD45 are both transmembrane phosphatases, these enzymes manifest their phosphatase activity through a catalytic cytosolic domain. We have utilized X-ray coordinates of related phosphatases (RPTPalpha and RPTmu) and comparative protein modeling to obtain molecular models of the D1 catalytic domains of CD45 and LAR. The models were tested using established protocols and found to be comparable to low resolution X-ray structures. The structure obtained for LAR was compared with the recently reported X-ray structure. Both the CD45-D1 and LAR-D1 structures were then compared to and contrasted with PTP1B. The active site of pockets of the three enzymes were found to be very uniform in structure and charge distribution. Also, the gross surface topology around the active site was found to be somewhat similar for the 3 phosphatases. However, there were significant differences in surface topology, and, more importantly, large changes in surface charge distribution. The differences between the surface features of these enzymes provide an explanation for the selectivity of inhibition by a number of peptides.

Secondary structures and intramolecular interactions in fragments of the B-loops of naturally occurring analogs of epidermal growth factor.

Structures of naturally occurring analogs of the B-loop fragment of human epidermal growth factor-like (hEGF-like) polypeptides were examined by molecular dynamics simulation in order to predict their secondary structures, to find structural similarity and to detect any weakly polar aromatic-aromatic (pi-pi) or amide-aromatic (N-pi) interactions which stabilize the structures. NPT molecular dynamics simulations (1 ns) were performed by the GRO-MACS package with SPC/E water using a weak temperature and pressure coupling method. During the sampling time, the structures of all peptides showed a characteristic secondary structure with a turn and bend at residues 4-7, and a beta-sheet, beta-bridge and random coil at the N- and C-terminal regions. Though the peptide chains were flexible, the stabilization effect of the N-pi interactions was indicated in some cases by the angles and distances between the centroids of aromatic planes of the side-chains and the H-atom of peptide bonds and the planes of the aromatic side-chains, respectively. Pi-pi interactions occurred less frequently because of the flexibility of the short peptide chain.

Isolation of multiple biologically and chemically diverse species of epidermal growth factor.

We have analyzed several lots of epidermal growth factor (EGF) purified from murine submaxillary glands including "receptor grade" EGF from Collaborative Research and EGF from Boehringer Mannheim Biochemicals. New England Nuclear uses "receptor grade" EGF to produce 125I-labeled EGF. Though these reagents are reported to be homogeneous, we found them to be a mixture of six species. A method was developed to separate this mixture into its component parts. The individual components were chemically characterized and tested for biological potency. N-terminal sequence analysis of the unfractionated EGF-mixture reveals three different sequences starting with residues 1, 2, or 3 of the mature peptide. Each component exhibited different degrees of mitogenic and EGF receptor binding activity indicating that the N-terminal region contributes to the biological response. The species representing the complete EGF peptide is the most active species in all biological assays. A rapid method for purification of homogeneous complete EGF from commercial EGF preparations is described.

A synthetic approach to structure-function relationships in the murine epidermal growth factor molecule.

Murine epidermal growth factor, a 53-amino acid peptide that is mitogenic for a number of cell types, has been synthesized by the solid-phase method. The synthetic peptide is identical to the natural material in amino acid composition, chromatographic behavior, receptor binding, and stimulation of DNA synthesis. Fragments of the EGF molecule corresponding to residues 42-53, 32-53, and 15-53 were constructed as well as the methionine sulfoxide derivative of EGF, [Met(O)21]EGF-(1-53), and a polymeric form of EGF. [Met(O)21]EGF-(1-53) was slightly less active than EGF in receptor binding and stimulation of DNA synthesis. Polymeric EGF was 1/100th as active as EGF, while EGF-(15-53) was less potent than EGF-(1-53) by a factor of 10(4). EGF-(32-53) was even less active and EGF-(43-53) was inactive.

EGF-ricin A conjugates: kinetic profiles of cytotoxic effects and resistant cell variants.

We have conjugated epidermal growth factor (EGF) with the A subunit of ricin (RICa) via a dithiopropionyl linkage. The EGF-RICa conjugate was competitive with [125I]EGF for binding to cell surface EGF receptors. Entry of the conjugate into cells was seen within 10 min at 37 degrees C and inhibition of protein synthesis was seen within 90 min. The blockage of protein synthesis continued for more than 20 h in sensitive cells. Protein synthesis in EGF receptor-deficient cells was not affected. The conjugate killed human epidermoid carcinoma (A431) cells, mouse 3T3 fibroblasts and Chinese hamster lung (CHL) cells with identical efficiencies (ED50 = 2 X 10(-9) M). The EGF-RICa conjugate was used as a selection agent and several resistant variants were isolated from CHL cells. These variants showed various degrees of [125I]EGF binding capacity. Some other characteristics of the variants are also described.