The Use of Phage-Displayed Peptide Libraries to Develop Tumor-Targeting Drugs

Monoclonal antibodies have been successfully utilized as cancer-targeting therapeutics and diagnostics, but the efficacies of these treatments are limited in part by the size of the molecules and non-specific uptake by the reticuloendothelial system. Peptides are much smaller molecules that can specifically target cancer cells and as such may alleviate complications with antibody therapy. Although many endogenous and exogenous peptides have been developed into clinical therapeutics, only a subset of these consists of cancer-targeting peptides. Combinatorial biological libraries such as bacteriophage-displayed peptide libraries are a resource of potential ligands for various cancer-related molecular targets. Target-binding peptides can be affinity selected from complex mixtures of billions of displayed peptides on phage and further enriched through the biopanning process. Various cancer-specific ligands have been isolated by in vitro, in vivo, and ex vivo screening methods. As several peptides derived from phage-displayed peptide library screenings have been developed into therapeutics in current clinical trials, which validates peptide-targeting potential, the use of phage display to identify cancer-targeting therapeutics should be further exploited

Identifying Drug Effects via Pathway Alterations using an Integer Linear Programming Optimization Formulation on Phosphoproteomic Data

Understanding the mechanisms of cell function and drug action is a major endeavor in the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomicbased approach to identify drug effects by monitoring drug-induced topology alterations. With the proposed method, drug effects are investigated under several conditions on a cell-type specific signaling network. First, starting with a generic pathway made of logical gates, we build a cell-type specific map by constraining it to fit 13 key phopshoprotein signals under 55 experimental cases. Fitting is performed via a formulation as an Integer Linear Program (ILP) and solution by standard ILP solvers; a procedure that drastically outperforms previous fitting schemes. Then, knowing the cell topology, we monitor the same key phopshoprotein signals under the presence of drug and cytokines and we re-optimize the specific map to reveal the drug-induced topology alterations. To prove our case, we make a pathway map for the hepatocytic cell line HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal Growth Factor Receptor (EGFR) and a non selective drug. We confirm effects easily predictable from the drugs’ main target (i.e. EGFR inhibitors blocks the EGFR pathway) but we also uncover unanticipated effects due to either drug promiscuity or the cell’s specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib is able to inhibit signaling downstream the Interleukin-1alpha (IL-1α) pathway; an effect that cannot be extracted from binding affinity based approaches. Our method represents an unbiased approach to identify drug effects on a small to medium size pathways and is scalable to larger topologies with any type of signaling perturbations (small molecules, 3 RNAi etc). The method is a step towards a better picture of drug effects in pathways, the cornerstone in identifying the mechanisms of drug efficacy and toxicity

DNA Display Selection of Peptide Ligands for a Full-Length Human G Protein-Coupled Receptor on CHO-K1 Cells

The G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II) type-1 receptor (hAT1R) as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO)-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells

PERSONALIZED IMMUNE DIAGNOSTICS: EPITOPE MAPPING OF THE IMMUNOME

Statistical phage display is a highly complex, but rapid and efficient way to identify “binding peptides” from a unique and specially designed library. It avoids repeated selection rounds and can therefore provide much more complex data than just a few sequences usually obtained with repeated peptide library selection. The complexity of the data analyzed is sufficient to cover hundreds of potential binder/target combinations in parallel.Applying this novel way to generate and analyze data from peptide phage display with antibodies allows to predict potential epitopes at amino acid resolution. Fingerprinting of monoclonal antibodies reveals the large variety of peptides binding to any given antibody. Independent of such laborious and failure prone methods like peptide arrays or phage display with antigen gene fragments. Surprisingly easy this can explain the specificity of antibodies and it is a valuable tool for antibody quality control.Beyond the application to individual antibodies we are able to analyze the immunome of patient sera. Theoretically, there are hundreds of antibody molecules for each recently encountered antigen epitope in a few µl. This is enough to define individual antibody epitopes. Since a single patient sample allows to record the entire immunome data, there is a tremendous amount of information hidden in the data sets we obtain. Nevertheless, all patients show different epitope patterns and for the generation of diagnostic tools we must compare many different sera. Results from examples will be given for allergic disease, viral infection diagnostics and the vaccine imprint on the immunome of one individual patient history.In infectious disease diagnostics (e.g. EBV, COVID-19, influenza) epitope-based kits can provide a robust analysis of existing and past disease as well as effective monitoring of vaccine efficacy. The aspect that the immune system carries the memories of antigens at least for many months allows a complex analysis even identifying different viral strains in a single experiment.In allergic disease we carried out epitope mapping with hundreds of sera from patients with sensibilization to allergenic food ingredients. Predicted epitopes were validated by binding IgE and IgG from many more patient sera for the main food allergy agents. Since peptide epitope diagnostics do not suffer from the undefined cross reactivities of present methods, we are gathering now a rather different understanding of what food allergies really are. In particular, we can also use IgG measurement based on immunoassays with epitopes, which has been regarded as impossible.Presently we are extending our work also in auto-immune diseases connected to long-COVID and psychiatric diseases

Functional Interrogation of the Kinome Using Nucleotide Acyl Phosphates

ABSTRACT: The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling. Protein kinases represent the single largest mammalian enzyme family with more than 500 members in the human proteome. These enzymes have been implicated in a wide array of complex cellular functions and pathways, ranging from metabolic regulation to tumorigenesis. Assessing kinase function in vivo is complicated by a high degree of posttranslational regulation, generally low expression levels, and overlapping substrate selectivity. Thus, despite intense efforts, the physiological function of the majority of protein kinases remains unknown. Considerable effort in the pharmaceutical industry is currently directed at the generation of novel protein kinase inhibitors (1). Several kinase inhibitors have been approved for clinical indications, including the anticancer drugs Gleevec (2) and Iressa (3). These drugs, as well as the majority of other kinase inhibitors in development, are designed to bind to the kinase ATP-binding site, thereby preventing substrate phosphorylation. Since the ATP-binding sites of protein kinases are highly conserved, the identification of inhibitors that are both potent and selective is paramount. At present, even the most comprehensive kinase selectivity screens cover less than 30% of the predicted protein kinases, leaving the majority of kinases unexplored. Recent reports have demonstrated that unexpected activities of protein kinase inhibitors can be found in kinase families structurally remote from the primary target (4, 5), highlighting the need for comprehensive screening. Additionally, due to the high degree of post-translational regulation of kinase activity, it is not clear whether assays of recombinant enzymes address the physiologically relevant state of each kinase. These observations, and the fact that many other families of ATP binding proteins exist with potential affinity for protein kinase inhibitors, highlight the need for improved methods for the comprehensive screening of protein kinases and other ATP binding proteins. Here we describe a probe-based technology (6, 7) that is uniquely capable of profiling the selectivity of kinase inhibitors against a broad range of protein kinases and other nucleotide binding proteins directly in native proteomes. This method also enables researchers to identify protein kinases and other ATPases with altered expression or activity in human disease or cell models. The method is based on novel biotinylated acyl phosphates of ATP or ADP that irreversibly react with protein kinases on conserved lysine residues in the ATP binding pocket. To date, more than 400 different protein kinases (>80% of the predicted kinome) have been identified and, in most cases, functionally assayed in various mammalian tissues and cell lines using this method. MATERIALS AND METHODS Synthesis of (+)-Biotin-Hex-Acyl-ATP (BHAcATP). 1 To a stirred suspension of N-(+)-biotinyl-6-aminohexanoic acid (30 mg, 0.085 mmol) in 3 mL of a dioxane/DMF/DMSO mixture (1:1:1) were added triethylamine (47 µL, 0.34 mmol) and isobutyl chloroformate (33 µL, 0.255 mmol) at 0°C. The cloudy mixture was kept at that temperature for 20 min, allowed to warm up to room temperature, and then stirred for an additional 1.5 h. A solution of ATP triethylammonium salt (69 mg, 0.085 mmol) in anhydrous DMSO (1 mL) was added to the mixture described above to give a clear solution. The reaction was monitored by 31 P NMR and MALDI. After 18 h, the reaction was quenched with water (4 mL) and the solution quickly extracted with ethyl acetate (3 × 4 mL)

What more do we want out of hair and from whom?

At the age of non-invasive approach to regenerative as well as other treatments, harmless yield of starting cell material plays a crucial role in the development of a therapy. The small sample size, absence of pain, bleeding, wound or any kind of postoperative complications can make all the difference in acceptance of the progressing therapy by regulatory bodies, medical practitioners and patients. Within such concept – particularly when it comes to autologous treatments – the importance of the mentioned benign sources grows and gains focus. Perhaps paradoxical in the venture-gain sense, such source appears within a tiny hair follicle, or more precise its outer root sheath (ORS). This is, to the best of our knowledge, the smallest source of adult stem cells, among which are, at that, the naivest cells of the adult human body, able to give raise to many cell types. Those ‘mother of all stem cells’ as they are called, present the closest match to embryonic stem cells in terms of their developmental potency. Potential of this compact stem cell pool to differentiate into neurons, glia, melanocytes, keratinocytes, fibroblasts, chondrocytes, osteocytes, cardiomyocites, and endothelial cells has already been shown. Mainly, our group at the Translational Centre for Regenerative Medizin / Dermatological Clinic At Leipzig University deals with the ectodermal and mesenchymal developmental potential of the ORS stem cell pool. We are currently focused on development of melanocytes and keratinocytes out of ORS for the purposes of non-invasive, autologous, transplantation-based therapies for wound healing and depigmentation, in particular Vitiligo. Standardized procedures have already been developed for quick, reliable and high yield cultivation of well defined, uniform primary cells available in antibiotic free sterile cultures based on explant culture of harmlessly plucked hair follicles. These procedures involve outgrowth on nylon meshes, favorable cultivation conditions for melanocytes, selection into pure culture and end characterization on protein and gene expression level. Those cells have been tested within a number of biocompatible scaffolds and the procedures for the cultivation and application have been patented. In cooperation with Fraunhofer Institute for Cell Therapy and Immunology, enzymatic modifications of scaffold materials by lacase have brought about more adhesive and friendlier conditions for the melanocytes and keratinocytes and with use of cafeic acid and L-DOPA as substrates granted us very promising candidates for graft carrier. Using similar procedures, we have cultivated cells that closely match mesenchymal stem cells out of human hair follicle ORS for the purposes of chondrogenic and osteogenic differentiation, aiming at regenerative therapies of cartilage and bone. Further on, the procedures have been adjusted to physiologically fit cultivation conditions for explant cultures other than human – horse, chimpanzee and rabbit. This abstract outlines not only our work in the field of ORS stem cells, but also that of others and comments on the entire vast regenerative potential of this elegant, nevertheless putative source

The active site of cellobiohydralase Ce16A from Trichorderma reesei: the roles of aspartic acids D221 and D175

Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of D2O(V) of 1.16 ( 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis