Synthesis, 3D-structure and stability analyses of NRPa-308, a new promising anti-cancer agent.

We report herein the synthesis of a newly described anti-cancer agent, NRPa-308. This compound antagonizes Neuropilin-1, a multi-partners transmembrane receptor overexpressed in numerous tumors, and thereby validated as promising target in oncology. The preparation of NRPa-308 proved challenging because of the orthogonality of the amide and sulphonamide bonds formation. Nevertheless, we succeeded a gram scale synthesis, according to an expeditious three steps route, without intermediate purification. This latter point is of utmost interest in reducing the ecologic impact and production costs in the perspective of further scale-up processes. The purity of NRPa-308 has been attested by means of conventional structural analyses and its crystallisation allowed a structural assessment by X-Ray diffraction. We also reported the remarkable chemical stability of this molecule in acidic, neutral and basic aqueous media. Eventually, we observed for the first time the accumulation of NRPa-308 in two types of human breast cancer cells MDA-MB231 and BT549.

Colorimetric immunoassays for the screening and specificity evaluation of molecules disturbing VEGFs/VEGFRs interactions.

Angiogenesis and its involved proteins, particularly Vascular Endothelial Growth Factor family (VEGFs) and VEGF receptors (VEGFRs), have been considered as a target of therapeutic interest for numerous inflammatory and vascular diseases. Acting on this biological process through interaction with VEGFs or VEGFRs has received considerable attention. Indeed, VEGFs and VEGFRs are currently targeted by drugs such as monoclonal antibodies. The feasibility of a therapeutic strategy based on blocking the VEGF/VEGFR interaction by using ligands "other-than-biologics" is also explored. To help to the discovery of new molecules, screening assays have been developed, particularly to evaluate the VEGFA/VEGFR1 interaction. Despite the therapeutic importance of VEGFB and PlGF (Placental Growth Factor), no assays have been developed to evaluate molecules against their interactions with VEGFR1. Here, we present new versatile colorimetric immunoassays to screen and evaluate the specific interaction of discovered molecules with different growth factors (VEGFA, VEGFB, PlGF) and receptors (VEGFR1, VEGFR2). These tests, based on competitive immunoassay format, will provide essential information on specificity and selectivity of molecules for their targets and will help to work on the pharmaco-modulation of molecules for targeting one specific interaction.

Synthesis and characterization of water-soluble macrocyclic peptides stabilizing protein α-turn.

Short peptides composed of naturally occurring amino acids are usually unstructured in aqueous media. The installation of covalent constraints within their side chains or backbones, resulting in the formation of macrocyclic peptides, is an appealing approach to stabilize them in defined secondary structures. Therefore, with the objective to stabilize α-turn conformation, we designed, synthesized and characterized constrained 13-membered macrocyclic peptides. Their design was inspired by previous work using the replacement of a hydrogen bond by a covalent bond, for the stabilization of α-helical secondary structures. Their synthesis employed our recently published solid-phase method based on Fukuyama-Mitsunobu alkylation reactions. We report herein an optimized synthesis leading to three water-soluble 13-membered macrocyclic peptides 10a-c, including respectively two, one and zero glycine residues. They were characterized by CD and NMR, which indicated the presence of equilibrating conformers. The detailed conformational analysis was based on extensive NMR and molecular dynamics studies. We found that the peptide without glycine residues 10c was mostly present as slowly interconverting conformers whereas the peptide with two glycine residues 10a was mostly present as rapidly interconverting conformers. We did not find a good match between the conformers of 10a and α-turns occurring in proteins, due to the high flexibility of the glycine backbone. Interestingly, we found that the major conformer of 10c accurately matched the "non-classical" or "tight" α-turn of type II-αLS, with a RMSD value of 0.42 Å for heavy atoms constituting the macrocycle. This is, to the best of our knowledge, the first molecule reported to mimic this type of α-turn found in proteins.

Targeted unlabeled multiple reaction monitoring analysis of cell markers for the study of sample heterogeneity in isolated rat brain cortical microvessels.

Liquid chromatography coupled to tandem mass spectrometry-based targeted absolute protein quantification (in fmol of the analyte protein per µg of total protein) is employed for the molecular characterization of the blood-brain barrier using isolated brain microvessels. Nevertheless, the heterogeneity of the sample regarding the levels of different cells co-isolated within the microvessels and bovine serum albumin (BSA) contamination (from buffers) are not always evaluated. We developed an unlabeled targeted liquid chromatography coupled to tandem mass spectrometry method to survey the levels of endothelial cells (ECs), astrocytes, and pericytes, as well as BSA contaminant in rat cortical microvessels. Peptide peak identities were evaluated using a spectral library and chromatographic parameters. Sprague-Dawley rat microvessels obtained on three different days were analyzed with this method complemented by an absolute quantification multiple reaction monitoring method for transporter proteins P-gp, Bcrp, and Na+ /K+ ATPase pump using stable isotope labeled peptides as internal standard. Inter-day differences in the cell markers and BSA contamination were observed. Levels of cell markers correlated positively between each other. Then, the correlation between cell marker proteins and transporter proteins was evaluated to choose the best EC marker protein for protein quantification normalization. The membrane protein Pecam-1 showed a very high correlation with the EC-specific transporter P-gp (Pearson product-moment correlation coefficient (r) > 0.89) and moderate to high with Bcrp (r ≥ 0.77), that can be found also in pericytes and astrocytes. Therefore, Pecam-1 was selected as a marker for the normalization of the quantification of the proteins of endothelial cells.

Synthesis and structure-activity relationship of non-peptidic antagonists of neuropilin-1 receptor.

Neuropilins (NRPs) are VEGF-A165 co-receptors over-expressed in tumor cells, and considered as targets in angiogenic-related pathologies. We previously identified compound 1, the first non-peptidic antagonist of the VEGF-A165/NRP binding, which exhibits in vivo anti-angiogenic and anti-tumor activities. We report here the synthesis and biological evaluations of new antagonists structurally-related to compound 1. Among these molecules, 4a, 4c and 4d show cytotoxic effects on HUVEC and MDA-MB-31 cells, and antagonize VEGF-A165/NRP-1 binding. This study confirmed our key structure-activity relationships hypothesis and paved the way to compound 1 'hit to lead' optimization.

Design and synthesis of C-terminal modified cyclic peptides as VEGFR1 antagonists.

Previously designed cyclic peptide antagonist c[YYDEGLEE]-NH2 disrupts the interaction between vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). It represents a promising tool in the fight against cancer and age-related macular degeneration. We described in this paper the optimization of the lead peptide by C-terminal modification. A new strategy for the synthesis of cyclic peptides is developed, improving the cyclisation efficiency. At 100 µM, several new peptides with an aromatic group flexibly linked at C-terminal end showed significantly increased receptor binding affinities in competition ELISA test. The most active peptide carrying a coumarin group may be a useful tool in anti-angiogenic biological studies.

Genome-wide scan for familial nasopharyngeal carcinoma reveals evidence of linkage to chromosome 4.

Nasopharyngeal carcinoma (NPC) occurs with high frequency in Asian populations, especially among people of Cantonese ancestry. In areas with high incidence, NPC clusters in families, which suggests that both geography and genetics may influence disease risk. Although the HLA-Bw46 locus is associated with increased risk of NPC, no predisposing genes have been identified so far. Here we report the results of a genome-wide search carried out in families at high risk of NPC from Guangdong Province, China. Parametric analyses provide evidence of linkage to the D4S405 marker on chromosome 4 with a logarithm of odds for linkage (lod) score of 3.06 and a heterogeneity-adjusted lod (hlod) score of 3.21. Fine mapping with additional markers flanking D4S405 resulted in a lod score of 3.54 and hlod score of 3.67 for the region 4p15.1-q12. Multipoint nonparametric linkage analysis gives lod scores of 3.54 at D4S405 (P = 5.4 x 10(-5)) and 4.2 at D4S3002 (P = 1.1 x 10(-5)), which is positioned 4.5 cM away from D4S405. When Epstein Barr virus antibody titer was included as a covariate, the lod scores reached 4.70 (P = 2.0 x 10(-5)) and 5.36 (P = 4.36 x 10(-6)) for D4S405 and D4S3002, respectively. Our findings provide evidence of a major susceptibility locus for NPC on chromosome 4 in a subset of families.

Recent advances of anti-angiogenic inhibitors targeting VEGF/VEGFR axis.

Vascular endothelial growth factors (VEGF), Vascular endothelial growth factor receptors (VEGFR) and their downstream signaling pathways are promising targets in anti-angiogenic therapy. They constitute a crucial system to regulate physiological and pathological angiogenesis. In the last 20 years, many anti-angiogenic drugs have been developed based on VEGF/VEGFR system to treat diverse cancers and retinopathies, and new drugs with improved properties continue to emerge at a fast rate. They consist of different molecular structures and characteristics, which enable them to inhibit the interaction of VEGF/VEGFR, to inhibit the activity of VEGFR tyrosine kinase (TK), or to inhibit VEGFR downstream signaling. In this paper, we reviewed the development of marketed anti-angiogenic drugs involved in the VEGF/VEGFR axis, as well as some important drug candidates in clinical trials. We discuss their mode of action, their clinical benefits, and the current challenges that will need to be addressed by the next-generation of anti-angiogenic drugs. We focus on the molecular structures and characteristics of each drug, including those approved only in China.

Identification of a human estrogen receptor α tetrapeptidic fragment with dual antiproliferative and anti-nociceptive action.

The synthetic peptide ERα17p (sequence: PLMIKRSKKNSLALSLT), which corresponds to the 295-311 region of the human estrogen receptor α (ERα), induces apoptosis in breast cancer cells. In mice and at low doses, it promotes not only the decrease of the size of xenografted triple-negative human breast tumors, but also anti-inflammatory and anti-nociceptive effects. Recently, we have shown that these effects were due to its interaction with the seven-transmembrane G protein-coupled estrogen receptor GPER. Following modeling studies, the C-terminus of this peptide (sequence: NSLALSLT) remains compacted at the entrance of the GPER ligand-binding pocket, whereas its N-terminus (sequence: PLMI) engulfs in the depth of the same pocket. Thus, we have hypothesized that the PLMI motif could support the pharmacological actions of ERα17p. Here, we show that the PLMI peptide is, indeed, responsible for the GPER-dependent antiproliferative and anti-nociceptive effects of ERα17p. By using different biophysical approaches, we demonstrate that the NSLALSLT part of ERα17p is responsible for aggregation. Overall, the tetrapeptide PLMI, which supports the action of the parent peptide ERα17p, should be considered as a hit for the synthesis of new GPER modulators with dual antiproliferative and anti-nociceptive actions. This study highlights also the interest to modulate GPER for the control of pain.

Association mapping identifies loci and candidate genes for grain-related traits in spring wheat in response to heat stress.

Heat stress is a limiting factor in wheat production along with global warming. Development of heat-tolerant wheat varieties and generation of suitable pre-breeding materials are the major goals in current wheat breeding programs. Our understanding on the genetic basis of thermotolerance remains sparse. In this study, we genotyped a collection of 211 core spring wheat accessions and conducted field trials to evaluate the grain-related traits under heat stress and non-stress conditions in two different locations for three consecutive years. Based on SNP datasets and grain-related traits, we performed genome-wide association study (GWAS) to detect stable loci related to thermotolerance. Thirty-three quantitative trait loci (QTL) were identified, nine of them are the same loci as previous studies, and 24 are potentially novel loci. Functional candidate genes at these QTL are predicted and proved to be relevant to heat stress and grain-related traits such as TaELF3-A1 (1A) for earliness per se (Eps), TaHSFA1-B1 (5B) influencing heat tolerance and TaVIN2-A1 (6A) for grain size. Functional markers of TaELF3-A1 were detected and converted to KASP markers, with their function and genetic diversity being analyzed in the natural populations. In addition, our results unveiled favor alleles controlling agronomic traits and/or heat stress tolerance. In summary, we provide insights into heritable correlation between yield and heat stress tolerance, which will accelerate the development of new cultivars with high and stable yield of wheat in the future.

A Cyclic Peptide Epitope of an Under-Explored VEGF-B Loop 1 Demonstrated In Vivo Anti-Angiogenic and Anti-Tumor Activities.

Pathological angiogenesis is mainly initiated by the binding of abnormal expressed vascular endothelial growth factors (VEGFs) to their receptors (VEGFRs). Blocking the VEGF/VEGFR interaction is a clinically proven treatment in cancer. Our previous work by epitope scan had identified cyclic peptides, mimicking the loop 1 of VEGF-A, VEGF-B and placental growth factor (PlGF), inhibited effectively the VEGF/VEGFR interaction in ELISA. We described here the docking study of these peptides on VEGFR1 to identify their binding sites. The cellular anti-angiogenic activities were examined by inhibition of VEGF-A induced cell proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). The ability of these peptides to inhibit MAPK/ERK1/2 signaling pathway was examined as well. On chick embryo chorioallantoic membrane (CAM) model, a cyclic peptide named B-cL1 with most potent in vitro activity showed important in vivo anti-angiogenic effect. Finally, B-cL1 inhibited VEGF induced human gastric cancer SGC-7901 cells proliferation. It showed anti-tumoral effect on SGC-7901 xenografted BALB/c nude mouse model. The cyclic peptides B-cL1 constitutes an anti-angiogenic peptide drug lead for the design of new and more potent VEGFR antagonists in the treatment of angiogenesis related diseases.

Two-step synthesis of a single-layer grafting self-floating adsorbent for anionic dyes adsorption, surface separation and concentration.

In the case of a sharp increase in the price of dyes, the dyes concentration of in wastewater is important for environmental protection and industrial costs reduction. In this study, we grafted single-layer amino groups onto the surface of the hollow glass microspheres by a two-step simple synthesis, and the anionic dyes adsorption process reaches equilibrium within 10 min. The as-synthesized adsorbent has self-floating ability to achieve high-efficiency surface solid-liquid separation with water. SEM, EDS, SBET, FT-IR, XPS, TGA characterizations results demonstrated the successful grafting of amino groups and the important role of the pretreatment process in the two-step synthesis. The results of adsorption isotherms and kinetics show the adsorption process belongs to single-layer adsorption with equal adsorption sites, and the adsorption capacities for acid orange 7 and amaranth reach 428.99 mg g-1 and 145.62 mg g-1, respectively. Regeneration of the adsorbent and concentration of the dye solutions can be achieved by dispersing the separated adsorbent in alkaline solution, the maximum concentrated concentration of acid orange 7 and amaranth was 6321.57 mg L-1 and 2431.84 mg L-1, respectively. This study provides new insights for the solid-liquid separation of water treatment agents and the resource regenerating of dyeing wastewater.

One-pot oxime ligation from peptides bearing thiazolidine and aminooxyacetyl groups.

One-pot oxime ligation under mild conditions using Pd(ii) as a shared catalyst from an aldehyde precursor (Thz) and a protected aminooxyacetyl group (Proc-Aoa) is reported. Two complementary metal-free protocols using unmasked Aoa-peptide are also described. Acetoxime-peptide can proceed to the desired oxime through an additional transoximation step.

Structural studies of the binding of an antagonistic cyclic peptide to the VEGFR1 domain 2.

Physiological and pathological angiogenesis is mainly regulated by the binding of the vascular endothelial growth factor (VEGF) to its receptors (VEGFRs). Antagonists of VEGFR are very attractive for the treatment of diseases related to excessive angiogenesis. Our previously designed C-terminal alkylated cyclic peptides [YKDEGLEE]-NHR (R = alkyl, arylalkyl) disrupt the interaction between VEGF and VEGFRs in biological assays. In this paper, we described the structural studies of the binding of one of these cyclic peptides named Peptide 3 to the VEGFR1 domain 2 (VEGFR1-D2). The molecular docking and NMR mapping identified the binding site on VEGFR1-D2. The anti-angiogenic effect of our peptide was evaluated by an experiment of VEGF-induced tube formation in two cell lines, retinal cell type RF6/A and vascular endothelial cell type HUVEC. Some new peptides were also synthesized and compared by an ELISA-based assay, in order to verify their ability to disrupt the formation of the complex VEGF-A/VEGFR1. In conclusion, the structural studies of Peptide 3 with VEGFR1-D2 will help the design of more efficient VEGFR antagonists. Moreover, Peptide 3, with improved receptor binding affinity, could be more suitable for VEGFR targeting bioimaging studies once labeled.

LC-MS/MS-based quantification of efflux transporter proteins at the BBB.

Targeted protein quantification using tandem mass spectrometry coupled to high performance chromatography (LC-MS/MS) has been used to quantify proteins involved in the absorption, distribution, metabolism and excretion (ADME) of xenobiotics to better understand these processes. At the blood-brain barrier (BBB), these proteins are particularly important for the maintenance of brain homeostasis, but also regulate the distribution of therapeutic drugs. Absolute quantification (AQUA) is achieved by using stable isotope labeled surrogate peptides specific to the target protein and analyzing the digested proteins in a triple-quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode to achieve a high specificity, sensitivity, accuracy and reproducibility. The main objective in this work was to develop and validate an UHPLC-MS/MS method for quantification of the ATP-binding cassette (ABC) transporter proteins Bcrp and P-gp and Na+/K + ATPase pump at the BBB. Three isoforms of the α-subunit from this pump (Atp1a 1, 2 and 3) were quantified to evaluate the presence of non-endothelial cells in the BBB using one common and three isoform-specific peptides; while Bcrp ad P-gp were quantified using 2 and 3 peptides, respectively, to improve the confidence on their quantification. The protein digestion was optimized, and the analytical method was comprehensively validated according to the American Food and Drug Administration Bioanalytical Method Validation Guidance published in 2018. Linearity across four magnitude orders (0.125 to 510 pmol·mL-1) sub-pmol·mL-1 LOD and LOQ, accuracy and precision (deviation < 15% and CV < 15%) were proven for most of the peptides by analyzing calibration curves and four levels of quality controls in both a pure solution and a complex matrix of digested yeast proteins, to mimic the matrix effect. In addition, digestion performance and stability of the peptides was shown using standard peptides spiked in a yeast digest or mouse kidney plasma membrane proteins as a study case. The validated method was used to characterize mouse kidney plasma membrane proteins, mouse brain cortical vessels and rat brain cortical microvessels. Most of the results agree with previously reported values, although some differences are seen due to different sample treatment, heterogeneity of the sample or peptide used. Importantly, the use of three peptides allowed the quantification of P-gp in mouse kidney plasma membrane proteins which was below the limit of quantification of the previously NTTGALTTR peptide. The different levels obtained for each peptide highlight the importance and difficulty of choosing surrogate peptides for protein quantification. In addition, using isoform-specific peptides for the quantification of the Na+/K + ATPase pump, we evaluated the presence of neuronal and glial cells on rat and mouse brain cortical vessels in addition to endothelial cells. In mouse liver and kidney, only the alpha-1 isoform was detected.

Development of binding assays for the SH2 domain of Grb7 and Grb2 using fluorescence polarization.

Adaptor proteins Grb7 and Grb2 have been implicated as being 2 potential therapeutic targets in several human cancers, especially those that overexpress ErbB2. These 2 proteins contain both a SH2 domain (Src homology 2) that binds to phosphorylated tyrosine residues contained within ErbB2 and other specific protein targets. Two assays based on enzyme-linked immunosorbent assay and fluorescence polarization methods have been developed and validated to find and rank inhibitors for both proteins binding to the pY(1139). Fluorescence polarization assays allowed the authors to determine quickly and reproducibly affinities of peptides from low nanomolar to high micromolar range and to compare them directly for Grb7 and Grb2. As a result, the assays have identified a known peptidomimetic Grb2 SH2 inhibitor (mAZ-pTyr-(alphaMe)pTyr-Asn-NH(2)) that exhibits the most potent affinity for the Grb7 SH2 domain described to date.

NRPa-308, a new neuropilin-1 antagonist, exerts in vitro anti-angiogenic and anti-proliferative effects and in vivo anti-cancer effects in a mouse xenograft model.

Neuropilin-1 (NRP-1) is an extra-cellular receptor for the main Vascular Endothelial Growth Factor over-expressed in tumour tissues, VEGF-A165. Consequently, NRP-1 is involved in angiogenesis and in tumour growth, and its over-expression is related to a clinical poor prognosis. NRP-1 appears as a major target in oncology, which remains poorly exploited. Herein, we report a new series of 18 small-sized fully organic VEGF-A165/NRP-1 antagonists (NRPas). These compounds share an original scaffold, including two linkers (sulphonamide and amide) and three aromatic cores. Among them, 2a (renamed NRPa-308) emerges as a promising "hit". In vitro,2a exerts not only potent anti-angiogenic activity, but also significant effects on cell viability of large panel of human solid and haematological cancer cell lines. Importantly, 2a is less cytotoxic on healthy tissues than the marketed anti-angiogenic drug sunitinib. Lastly, in a mouse xenograft model (human MDA-MB-231 breast cancer cells), 2a improves the median survival and reduces the tumour growth, but does not exert visible acute toxicity. Altogether, these results highlight its huge potential for a further "hit-to-lead" optimization, leading to new anti-cancer drugs.

Identification of Peptidic Antagonists of Vascular Endothelial Growth Factor Receptor 1 by Scanning the Binding Epitopes of Its Ligands.

Cancer angiogenesis is mainly initiated by vascular endothelial growth factors (VEGFs). On the basis of the reported crystal structures of three natural ligands (VEGF-A, -B, and PlGF) with the major receptors VEGFR-1 and VEGFR-2, we scanned receptor-binding epitopes of these ligands by designing linear and cyclic peptides with the aim to disrupt the VEGF-A/VEGFR-1 interaction, which is implicated in cancer development. The ability of peptides to inhibit this interaction was evaluated by an ELISA-based assay. Several peptides, especially those mimicking loop 1 (L1) of these ligands that binds primarily to domain D3 of VEGFRs, have demonstrated higher inhibition for VEGF-A/VEGFR-1 binding. They have also shown inhibitory effects on VEGF-induced tube formation in HUVECs (human umbilical vein endothelial cells). These results validate the domain D3 of VEGFRs as an efficient target for the design of VEGFR antagonists.

[18F]MEL050 as a melanin-targeted PET tracer: Fully automated radiosynthesis and comparison to 18F-FDG for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases.

INTRODUCTION: Melanoma is a highly malignant cutaneous tumor of melanin-producing cells. MEL050 is a synthetic benzamide-derived molecule that specifically binds to melanin with high affinity. Our aim was to implement a fully automated radiosynthesis of [18F]MEL050, using for the first time, the AllInOne™ synthesis module (Trasis), and to evaluate the potential of [18F]MEL050 for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases, and to compare it with that of [18F]FDG. METHODS: Automated radiosynthesis of [18F]MEL050, including HPLC purification and formulation, were performed on an AllInOne™ synthesis module. [18F]MEL050 was synthesized using a one-step bromine-for-fluorine nucleophilic heteroaromatic substitution. Melanoma models were induced by subcutaneous (primary tumor) or intravenous (pulmonary metastases) injection of B16-F10-luc2 cells in NMRI mice. The maximum percentage of [18F]MEL050 Injected Dose per g of lung tissue (%ID/g Max) was determined on PET images, compared to [18F]FDG and correlated to in vivo bioluminescence imaging. RESULTS: The automated radiosynthesis of [18F]MEL050 required an overall radiosynthesis time of 48min, with a yield of 13-18% (not-decay corrected) and radiochemical purity higher than 99%. [18F]MEL050 PET/CT images were concordant with bioluminescence imaging, showing increased radiotracer uptake in all primary subcutaneous tumors and pulmonary metastases of mice. PET quantification of radiotracers uptake in tumors and muscles demonstrated similar tumor-to-background ratio (TBR) with [18F]MEL050 and [18F]FDG in subcutaneous tumors and higher TBR with [18F]MEL050 than with [18F]FDG in pulmonary metastases. CONCLUSION: We successfully implemented the radiosynthesis of [18F]MEL050 using the AllInOne™ module, including HPLC purification and formulation. In vivo PET/CT validation of [18F]MEL050 was obtained in mouse models of pigmented melanoma, where higher [18F]MEL050 uptake was observed in sub-millimetric pulmonary metastases, comparatively to [18F]FDG.

Crystal structures of the SH2 domain of Grb2: highlight on the binding of a new high-affinity inhibitor.

The activation of growth factor receptors induces phosphorylation of tyrosine residues in its C-terminal part, creating binding sites for SH2 domain-containing proteins. Grb2 is a protein that recruits Sos, the exchange factor for Ras. Recruitment of Sos allows for Ras activation and subsequent signal transmission. This promotes translocation of MAP kinases into the nucleus and activation of early transcription factors. Grb2, a 25 kDa protein, is composed of one SH2 domain surrounded by two SH3 domains. The SH2 domain of Grb2 binds to class II phosphotyrosyl peptides with the consensus sequence pYXNX. Thus, Grb2 is a good example of a bifunctional adaptor protein that brings proteins into close proximity, allowing signal transduction through proteins located in different compartments. To explore the interactions between Grb2 and phosphorylated ligands, we have solved the crystal structure of complexes between the Grb2-SH2 domain and peptides corresponding to Shc-derived sequences. Two structures are described: the Grb2-SH2 domain in complex with PSpYVNVQN at 1.5 A; and the Grb2-SH2 domain in complex with mAZ*-pY-(alphaMe)pY-N-NH2 pseudo-peptide, at 2 A. Both are compared to an unliganded SH2 structure determined at 2.7 A which, interestingly enough, forms a dimer through two swapping subdomains from two symmetry-related molecules. The nanomolar affinity of the mAZ-pY-(alphaMe)pY-N-NH2 pseudo-peptide for Grb2-SH2 is related to new interactions with non- conserved residues. The design of Grb2-SH2 domain inhibitors that prevent interaction with tyrosine kinase proteins or other adaptors like Shc or IRS1 should provide a means to interrupt the Ras signaling pathway. Newly synthesized pseudo-peptides exhibit nanomolar affinities for the Grb2-SH2 domain. It will then be possible to design new inhibitors with similar affinity and simpler chemical structures.