Gene regulatory peptides

Abstract

The invention relates to the modulation of gene expression in a cell, also called gene control, in particular in relation to the treatment of a variety of diseases. The invention provides a method for modulating expression of a gene in a cell comprising providing the cell with a signaling molecule comprising a peptide or functional analogue thereof. Furthermore, the invention provides a method for identifying or obtaining a signaling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing the cell with a peptide or derivative or analogue thereof and determining the activity and / or nuclear translocation of a gene transcription factor.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of PCT International Application Number PCT / NL02 / 00639, filed Oct. 4, 2002, and published in English as International Publication Number WO 03 / 029292 A2 on Apr. 10, 2003, which claimed continuation-in-part status from U.S. patent application Ser. No. 10 / 028,075, filed Dec. 21, 2001, the contents of all of which are incorporated herein by this reference.BACKGROUND OF THE INVENTION [0002] This invention relates generally to biotechnology, and more particularly to gene regulatory peptides and methods of their use. [0003] Gene control is generally thought to occur at four levels: 1) transcription (either initiation or termination), 2) processing of primary transcripts, 3) stabilization or destabilization of mRNAs, and 4) mRNA translation. The primary function of gene control in cells is to adjust the enzymatic machinery of the cell to its nutritional, chemical and physical environment. [0004] It is general...

AI Technical Summary

Benefits of technology

[0035] Synthetic peptides can be obtained using various procedures known in the art. These include solid phase peptide synthesis (SPPS) and solution phase organic synthesis (SPOS) technologies. SPPS is a quick and easy approach to synthesize peptides and small proteins. The C-terminal amino acid is typically attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products.

[0037] An important link in any polypeptide chain is the amide bond, which is formed by the condensation of an amine group of one amino acid and a carboxyl group of another. The replacement of key amide bonds in peptide fragments by isosteric groups has recently received considerable attention as a possible means of generating novel bio-active substances with improved stability. In one embodiment of the invention, a lead peptide comprises a synthetic molecule in which at least one amide bond has been replaced by an isosteric group such as a ketomethylene or a trans-alkene group. Classically, well-defined molecules were systematically modified and the product compounds analyzed for improved biological activity. Newer combinatorial chemistry methods allow the synthesis of a large population of similar compounds. This is generally followed by the selection, or screening, of peptides for biological activity such as the capacity to regulate gene expression. In one embodiment of the invention, lead peptides are synthesized in a random fashion using a combinatorial chemistry approach. Combinatorial chemistry, combined with recent advances in robotic screening, enables the testing of a large number of compounds in a short period of time. This technique involves the preparation of a large number of structurally related compounds either as mixtures in the same reaction vessel or individually by parallel synthesis. In this manner, large pools of similar compounds can be synthesized within a short period of time. Combinatorial libraries can be prepared using both solution chemistry and by solid phase synthesis; however, solid phase synthesis allows the use of excess reagents to drive the reaction to completion and easy removal of the reagents and side-products by simple filtration of the polymeric support and washing with solvent. Therefore, solid phase synthesis offers a more attractive approach to the generation of chemical libraries for screening purposes.

[0038] Combinatorial chemistry is well suited to peptides. Lead peptide libraries can be easily synthesized using solid-phase chemistry. Sequence degeneracy can be incorporated during the synthesis using either split synthesis or parallel synthesis. In the split synthesis approach, the solid support is divided into portions prior to each coupling step. A different molecular unit (synthon), like an amino acid, is then coupled to each portion. All portions are recombined after coupling and the synthesis cycle is completed. This “split and mix” approach has the advantage of yielding a unique sequence on each support bead and variability in synthon reactivity can be corrected by varying the coupling conditions. Peptide synthesis, where variations in reactivity between amino acids are significant, requires the “split and mix” approach. Head-to-tail cyclization of peptides on the resin provides a facile route to cyclic compounds. In addition to general advantages of solid phase synthesis, such as high efficiency and easy purification, head-to-tail cyclization of peptides on polymer supports provides minimal risk of intermolecular reactions (e.g., dimerization and oligomerization), even under high concentration. This is another advantage over solution chemistry which requires high dilution conditions to avoid intermolecular side reactions of the linear peptide.

[0048] Reporter gene assays allow analysis of a large number of different samples in a relatively short time. It can easily be performed using multiwell plates such as 96-well plates. The activity of many reporter genes can be assayed using colorimetric or fluorescence detection in, for example, an automated plate reader. Thus, a reporter gene assay can be used in a high-throughput format. This is especially advantageous when performing several rounds in screening for gene regulatory effects of a peptide, for example, in the process of lead peptide optimalization. In these types of assays, it is preferred to focus on a small number of different promotor elements. For example, cells can be transfected with a reporter gene construct for the detection of NFkappaB activity, or with a reporter gene construct for AP-1 activity or with a reporter construct designed to readily determine NFAT-1 activity. Cells can be transfected in parallel with one reporter gene construct but it is also possible to provide a cell with more than one reporter gene construct. Of course, to allow discrimination between activities of the different promoters, it is preferred that each promotor construct contains a different reporter gene. In one embodiment of the invention, a cell is provided with more than one reporter gene construct to determine the effect of a peptide or a derivative or analogue thereof on transcriptional activity. For example, a cell is cotransfected with two or even three different plasmids, each containing a distinct fluorescent reporter gene fused downstream of a distinct promotor of interest. From this, the effect of the peptide on each promotor is determined. In such an experimental setup, it is obviously preferred that reporter gene products of the cotransfected reporter constructs are easily distinguished from each other. Interesting reporter genes that can be used in cotransfection reporter gene assays include GFP or EGFP (enhanced green fluorescence protein) and spectral variants thereof, such as RFP, YFP and CFP. Following exposure of the cell to a peptide according to the invention, the activities of each fluorescent reporter gene is measured by fluorescence detection using a suitable optical filter set, like a multi-band filter set. Multi-band sets are used for multiple labeling and simultaneous viewing of multiple fluorophores. Each set of exciters, dichroics, and emitters yields isolated bands of excitation and emission energy.

[0050] Using a method for identifying or obtaining a signaling molecule comprising a peptide or functional derivative or analogue thereof capable of modulating expression of a gene in a cell comprising providing the cell with a peptide or derivative or analogue thereof and determining the activity and / or nuclear translocation of a gene transcription factor as provided herein furthermore allows, at random, testing of a multiplicity of oligo- or lead peptides, leading to automated combinatorial chemistry formats, wherein a great many of candidate signal molecules are being tested in a (if so desired, at random) pattern for their reactivity with a molecular library of synthetic peptides representing potential signal molecules allowing the rapid detection of particularly relevant molecules out of tens of thousands of (combinations of) molecules tested. In a preferred embodiment, the invention provides a method wherein the lead peptides, or at least their activities, are positionally or spatially addressable, e.g., in an array fashion, if desired aided by computer directed localization. In an array, the pluralities are, for example, addressable by their positions in a grid or matrix.

Problems solved by technology

These steps define the regulatory decisions in a transcriptional circuit and misregulation at any stage can result in a variety of diseases.

Some receptor-ligand interactions, however, are known to cause prompt nuclear transcriptional activation of a specific and limited set of genes.

However, progress has been slow in determining exactly how such activation is achieved.

These drugs are considered to be nonspecific and often only applicable in high concentrations that may end up toxic for the individual treated.

Under these conditions, it can be difficult to use the peptide in experiments, and it may be difficult to purify the peptide if necessary.

Peptides containing multiple Cys, Met, or Trp residues can also be difficult to obtain in high purity partly because these residues are susceptible to oxidation and / or side reactions.

Certain side chains contain functional groups that can interfere with the formation of the amide bond.

Also, it can lead to transcriptional repression of target genes.

Bone disease is often characterized by a disturbance in this balance such that there is a net increase in bone resorption over bone formation.

The white blood cells bound to the endothelium eventually migrate into the brain or cardiac tissue, causing significant tissue destruction.

Anthrax, the disease caused by the spore-forming Bacillus anthracis (B. anthracis), continues to be a worldwide problem among domesticated and wild herbivores in Asia and Africa and poses a worldwide threat when being used as biological weapons for biological warfare or bioterrorism.

Outbreaks or epidemics are a constant threat for endemic regions because spores can persist in the soil for long periods of time.

Therapeutic intervention in general must be initiated early, as septicemic infections are nearly always fatal.

As said, anthrax is a disease of animals and humans and poses a significant threat as an agent of biological warfare and terrorism.

Inhalational anthrax, in which spores of B. anthracis are inhaled, is almost always fatal, as diagnosis is rarely possible before the disease has progressed to a point where antibiotic treatment is ineffective.

In addition, high levels of lethal toxin lead to lysis of macrophages within a few hours by an unknown mechanism.

Recent data suggests that this happens due to inhibition of growth-factor pathways leading to macrophage death.

This may explain the symptoms before death which are characterized by the hyperstimulation of host macrophage inflammatory pathways, leading to dramatic hypotension and shock.

However, persistent activation of NF-kappaB may lead to the production of excessive amounts of pro-inflammatory mediators such as IL-12 and TNF-alpha, resulting in tissue damage, as in insulin-dependent diabetes mellitus, atherosclerosis, Crohn's disease, organ failure, and even death of the host, as in bacterial infection-induced septic shock.

The concentrations of an active molecule which can be administered would be limited by efficacy at the lower end and the solubility of the compound at the upper end.

In a further embodiment, such treatment also comprises the use of, for example, an antibiotic, however, only when such use is not contraindicated because of the risk of generating further toxin loads because of lysis of the bacteria subject to the action of those antibiotics in an individual thus treated.

Although a vaccine against anthrax exists, various factors make mass vaccination impractical.

The bacteria can be eradicated from the host by treatment with antibiotics, but because of the continuing action of the toxin, such therapy is of little value once symptoms have become evident.

In particular, erythroid and thromboid cells are susceptible to the treatment.

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