WO2009068321A1 - Site-directed high throughput screening - Google Patents

Abstract

The present invention relates to the identification of binder compounds for a certain target by a site-directed screening method, whereby the binding of a compound can be detected in a parallel detection assay. The invention also relates to the modification of these compounds as well as the use in a pharmaceutically acceptable form.

Description

Site-directed high throughput screening

Background

The conventional approach to identify biologically active, drug-like small molecules is based on high-throughput screening of chemical libraries. However, the synthesis of large chemical libraries and their screening are time-consuming and expensive; the success relies heavily on the quality of the available libraries and even the largest library can span only a minute section of the virtual chemical space. Therefore, over the recent decade several strategies have been developed with the goal to use the protein target as a template for ligand assembly thus reducing the synthetic effort required for ligand development significantly.

Ligands can be directly identified from relatively small libraries of low molecular-weight fragments by employing nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography. These "fragment-based" methods usually deliver low-affinity ligands as rational starting points for iterative development of more potent molecules together with structural information on ligand binding; however, they rely on demanding technical prerequisites such as the availability of protein crystals or NMR structures, large amounts of protein or high protein solubility which limit the general applicability of these methods.

As an alternative to the "direct" methods, virtual combinatorial libraries, also called dynamic combinatorial libraries, have been introduced for protein ligand development. In this ap- proach, mixtures of reactive molecules form ligation products in a dynamic equilibrium reaction. When a protein is added as a template, some library constituents might bind to it and thereby are removed from the pool of interconverting compounds. As a result, the equilibrium is shifted in the presence of the protein template, resulting in an amplification of the good binders and a reduction of the concentration of poor binders in the library. The general prob- lem of virtual chemical libraries is the detection of the selected ligation products. In the original realization of the concept, imines as the ligation products were fixed chemically by reduction and analyzed by High performance liquid chromatography (HPLC). As alternatives, NMR and affinity chromatography were employed to identify the preferred ligands.

In a strategy called "tethering", natively or artificially introduced cysteine residues on the protein surface were reacted with a library of disulfide-containing small-molecule fragments. Fragments binding to protein sites adjacent to the cysteine reacted preferably under disul- fide-bond formation. Detection of the formed protein adducts resulting in a mass shift of the protein signal was carried out by Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The need for genetic modification of the target is one of the drawbacks of this method.

All reported fragment-based methods, "direct" or dynamic, have in common that the preferred protein ligands are selected solely by binding, not by biological activity. Only the tethering approach allows the selective identification of ligands that bind to discrete regions of the molecular target, whereas for the other methods, in principle, every sub-pocket of the macro- molecule can act as a binding site. Furthermore, despite of the indisputable progress made in the development of protein ligands, the reported fragment-based methods suffer from the mostly difficult, time-consuming, and expensive detection of active compounds, either by chromatography (LC/MS), X-ray crystallography, or NMR. Stoichiometric amounts of protein are required for all thermodynamically driven strategies such as those based on NMR, crys- tallography and dynamic combinatorial libraries. Few examples of kinetically driven selection have been reported.

Another problem of methods known in the state of art is the detection of the binding. So far there are not assays available allowing a simple detection. Separately performed detection bears the risk of false positive or false negative results, because additional steps are necessary. Therefore the detection of the binding of fragments to the target is a limiting factor for high-throughput assays.

Accordingly, there is a need for the development of a method for the high-throughput identifi- cation of fragments for a target, which is superior to the prior art and to further optimize these fragments via chemical modification.

Summary of the invention

The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.

Surprisingly the invention solves the above technical problem by providing a method comprising the following steps:

The invention relates to a method for a rapid and sensitive identification of binding and activity of low-affinity fragments for an active site of a target, wherein a formation of a binding- species comprising one of the said fragments and an electrophilic binder preferred via a reversible ligation reaction is detected in a parallel detection assay.

Thus the invention relates to the surprising discovery, that it is possible to identify low-affinity binders by the use of a site-directing probe and to detect the binding in parallel assay. In this context "site-directing" probe means that the electrophile binder binds to a defined site of the target, thus the electrophile is directing the compound (nucleophile) to this site, while forming a binding-species. Therefore the potential product formed (binding-species) in the ligation equilibrium can be directly evaluated for their biological activity towards a target. The identification method can be adapted to standard in-vitro protein assays, which is a major advan- tage compared to methods in the state of art.

The method of the invention allows testing of inhibitory fragments for a defined site on the target with high sensitivity and without the need for genetic modification of the target. Therefore fragments are identified faster compared to the state of art.

In this context a low-affinity fragment is a fragment that binds a target with a dissociation constant K₀ of more than 100 μM. Low-affinity binders are generally difficult to detect by methods known in the state of art. Most methods in the state of art are not sensitive enough to detect low-affinity binders. The method of the invention enables the identification of binding fragments with inhibition constants in the millimolar range, which is a great advantage compared to methods in the state of art.

The identification of low-affinity binder provides access to a larger group of possible binders, which alleviates the identification and the creation of optimal fragments for the binding to a target.

The low-affinity fragments identified by the method of the invention are not capable to bind the target by themselves. Therefore even an extremely sensitive method would not identify theses fragments as binders. The invention allows the identification of these fragments due to the use of an electrophilic binder.

The term "electrophile" is art-recognized and refers to chemical moieties which can accept a pair of electrons from a nucleophile. The electrophilic binder of the invention is selected from the group comprising aldehydes, ketones, carboxylic acid ester, carboxylic acid thioester, imines, iminium salts (ions), halo- genides, epoxides, elektrophilic olefins (,,Michael-acceptors"). The electrophilic binder binds to a defined site of the target, preferred an active site. The electrophilic binder can be designed based on a virtual screen of the target, for example using the docking program FlexX. The electrophilic binders of the invention are advantageous due to their good water solubility. Also the use of peptidic binders as electrophiles is advantageous. It is easier to design a peptidic binder for a defined site of a target, for example based on crystal structure information compared to non-peptidic binders. Peptidic binders are not very suitable for pharmaceutical uses; therefore it is preferred to use the method of the invention to optimize a peptidic binder to a non-peptidic inhibitor.

It is understood, that the invention also relates to a method wherein the site directing probe is a nucleophile and the low-affinity binders are selected from the group comprising electrophiles.

Table 1 : Alternatives for the electrophilic binder:

The term "target" is used in the broadest sense and refers to a chemical or biological entity. The target can be a molecule, a portion of a molecule, or an aggregate of molecules. Specific examples of target molecules include polypeptides (e.g., enzymes, receptors, transcription factors, ligands for receptors, growth factors, immunoglobulins, nuclear proteins, signal transduction components, allosteric enzyme regulators, and the like), polynucleotides, peptides, carbohydrates, glycoproteins, glycolipids, and other macromolecules, such as nucleic acid-protein complexes, chromatin or ribosomes, lipid bilayer-containing structures, such as membranes, or structures derived from membranes, such as vesicles.

An "active site" refers to a region of a target that, as a result of its shape and/or charge po- tential, favourably interacts or associates with another agent (including, without limitation, a protein, polypeptide, peptide, nucleic acid, including DNA or RNA, molecule, compound, antibiotic or drug) via various covalent and/or non-covalent binding forces. Examples for an active site in an enzyme are catalytic sites, substrate binding sites, cofactor binding sites or allosteric sites. Every site within a target, or on its surface, at which ligands can bind are active sites of the invention.

If the macromolecule is a nucleic acid, then binding sites may be the bases of the nucleic acid, or spaces in their structures, for example the major or minor grooves in the helical DNA, interactions with phosphate, ribose or deoxyribose groups or intercalated between the bases.

The term "binding" refers to an affinity between two molecules, for example, a compound and a target molecule. As used herein, "binding" means a preferential binding of one molecule for another in a mixture of molecules, e.g. with a binding affinity is about 1x10⁴ M to about 1x10⁶ M or greater.

The binding of a compound to a target molecule can be considered specific if the binding affinity to one target is significantly stronger than to another target, e.g. 2-3 times stronger, in the better case > 10 times stronger, in the optimal case > 50-100 times stronger.

In preferred embodiment the method is a method for a production of a binding-species and/or an identification of a binding of a compound and/or the binding-species to an active site of a target, wherein the method comprises the following steps:

a) providing the target,

b) addition of an identified and/or synthesized electrophilic binder, preferred selected from table 1 , especially an aldehyde which binds to the active site of the target,

c) addition of a first compound, selected from the group of low molecular weight compounds, in particular nucleophiles, preferred amines, secondary amines, isocyanides, thiols, alcohols, hydrazines, enolates, enolized aldehydes, enolized ketones, phosphorylides, hydrides, car- banions and/or hydroxylamines especially preferred selected from the group comprising the compounds of table 2, d) detection of the binding, preferred a reversible binding, of a first binding-species to the target in a parallel detection assay, wherein the first binding-species is a product of a ligation, preferred a reversible ligation, of the first compound and the electrophilic binder, and wherein the first compound and/or the first binding-species are identified and/or produced.

To realize the method of the invention, preferred in the first step a target is provided. Next a detection assay for the target will be developed. An electrophilic binder for the target will be selected as a directing probe; the electrophilic binder functionality will serve as reactive group for ligation of nucleophiles. A library of nucleophiles will be designed and tested in the presence of the ligation probe in the detection assay. Preferred primary hits will be confirmed by chemical synthesis and testing of analogs in order to verify the site-directed binding with the target and to optimize the binding affinity of the resulting non-peptidic inhibitor. The parallel detection (d) highly simplifies the identification of new compounds. The method of the invention is less time-consuming, cheaper and can be carried out without the need for expensive equipment.

The terms "fragment" and "compound" are used interchangeable.

The term "nucleophile" is recognized in the art, and as used herein means a chemical moiety having a reactive pair of electrons. The nucleophiles used in the invention are in compliance with physicochemical criteria, including the Lipinski rules. Preferably the compounds are selected by a diversity analysis and a subsequent pharmacophoric search using Unity (SYBYL 7.0, Tripos Inc., St. Louis, Missouri, 63144, USA). Unity is a search and analysis tool for exploring chemical databases. It is capable of finding molecules that satisfy user-defined que- ries based on molecular fragments, acceptor or donor sites on both molecule or receptor site constraints.

Table 2: low molecular weight compounds

The term "low-molecular weight" compounds relates to compounds with a small size, usually less than 500 Daltons, preferred less than 300 Daltons, especially preferred less than 250 Daltons. Identifying low-molecular weight compounds is advantageous because subsequent modification and optimization are easier compared with larger compounds.

The reversible binding to the target is benefiting for the use of these compounds in pharmaceutical compositions. Irreversible binding of compounds e.g. inhibitors often leads to various side-effects, so that the use of such compounds in pharmaceutical compositions is disadvantageous.

The method of the invention is superior to the state of art because there are no false positives. Nonspecific binding does not occur, because it is possible to only detect compounds that bind specifically in a regular fashion.

This method avoids disadvantages associated with screening methods in which the alteration of target activity by a potential ligand is assessed, as in that case compounds which bind weakly but non-specifically can alter macromolecule activity in a non-selective manner. Such non-selective inhibition produces false-positives, as the assay shows target inhibition, but the compound would perform no useful function as a drug, as it would interfere with the activity of other proteins as well. Only compounds bound in a defined binding site will be detected by the present method, which reduces the number of false-positives. The method of the invention includes a detection assay which is carried out parallel to the identification method. Therefore no extra steps, like changing medium or transferring the mixture in another reaction vessel are necessary. This is superior because contaminations or permutation of probes are prevented. Additionally the method is extremely time-saving, which also reduces the costs for the method. Less work steps are needed, compared to methods in the state of art, therefore the method is more convenient, cheaper and faster.

The possibility of detecting the binding in a parallel detection assay enables the performance of a high-throughput method, not only for the identification but also for the detection in one approach. The use of standard protein assays is preferred.

The method of the invention employs a biochemical assay that can be operated in high- throughput for the detection of active formation products. Thereby, the potential product formed in the ligation equilibrium can be directly evaluated for their biological activity towards a target. Moreover, if one formation partner (the electrophilic binder) is known to bind to a defined site this methodology allows the site-directed identification of binding fragments.

Also preferred is the method for an identification and/or production of a binder for an active site of a target, wherein the method comprises the following steps:

a) providing the target,

b) addition of an identified and/or synthesized electrophilic binder, preferred selected from table 1 , especially an aldehyde, which binds to the active site of the target,

c) addition of a first compound, selected from the group of low molecular weight compounds, in particular nucleophiles, preferred amines, secondary amines, isocyanides, thiols, alcohols, hydrazines, enolates, enolized aldehydes, enolized ketones, phosphorylides, hydrides, car- banions and/or hydroxylamines especially preferred selected from the group comprising the compounds of table 2,

d) detection of the binding, preferred a reversible binding, of a first binding-species to the target in a parallel detection assay, wherein the first binding-species is a product of a ligation, preferred a reversible ligation, of the first compound and the electrophilic binder, and wherein the binder is the first compound and/or the first binding-species.

Another preferred embodiment of the invention is the method, wherein the method comprises a modification of the identified first compound, preferred an introduction of an electrophilic moiety, especially preferred an electrophilic warhead whereby a modified second compound is obtained. Preferably the modified second compound is selected from the group comprising the compounds of table 1. It is preferred that the compounds identified by the methods of the present invention are subsequently modified to alter their binding to the target macromolecule or to improve their usefulness as a pharmaceutical.

These modifications are conventional in the art. Possible modifications include: substitution or removal of groups containing residues which interact with the target macromolecule, for example groups which interact with the amino acid side chain groups of a protein; the addition or removal of groups in order to alter the charge of a group in a compound; the replacement of a charge group with a group of the opposite charge; or the replacement of a hydrophobic group with a hydrophilic group or vice versa. Additionally, a group may be replaced with another retaining similar properties but that better occupies the cavity in the macromolecule increasing the surface of the ligand in contact with the macromolecule cavity. This may be achieved using by conventional synthetic approaches typically utilized by those skilled in the art of medicinal chemistry. Many of these changes will improve the usefulness of a compound as a pharmaceutical. It will be understood that these are only examples of the type of substitutions considered by medicinal chemists in the development of new pharmaceutical compounds and other modifications may be made, depending upon the nature of the starting compound and its activity.

The term "group" is used herein to refer to a portion of a molecule, typically having a particular functional or structural feature, e.g. a linking group (a portion of a molecule connecting two other portions of the molecule), or an ethyl moiety (a portion of a molecule with a structure closely related to ethane). A "group" includes both substituted and unsubstituted forms. Typical substituents include one or more lower alkyl, modified alkyl, any halogen, hydroxy, or aryl. Any substituents are typically chosen so as not to substantially adversely affect reaction yield (for example, not lower it by more than 20% (or 10%, or 5% or 1%) of the yield otherwise obtained without a particular substituent or substituent combination).

Surprisingly, it was possible to modify the identified low-affinity binder in a way it is able to bind the target itself, without a site-directing electrophilic.

A skilled artisan knows which kind of modification is necessary, according to the used electrophilic and the identified nucleophile.

Another preferred embodiment of the invention relates to the method further comprising the following steps:

e) providing the target,

f) addition of the modified second compound and/or a derivative thereof,

g) addition of the first compound and/or a third compound selected from the group of low molecular weight compounds, in particular nucleophiles, preferred amines, secondary amines, isocyanides, thiols, alcohols, hydrazines, enolates, enolized aldehydes, enolized ketones, phosphorylides, hydrides, carbanions and/or hydroxylamines especially preferred selected from the group comprising the compounds of table 2,

h) detection of a binding, preferred a reversible binding, of a second binding-species to the target in a parallel detection assay, wherein the second binding-species is a product of a ligation, preferred a reversible ligation, of the third compounds and/or the first compound and the modified second compound and/or a derivative thereof, and wherein the third compound and/or the first compound and/or the second binding-species are identified and/or produced, preferred wherein the second binding-species is non-peptidic, preferred selected from the group comprising the compounds of table 3.

Table 3: Possible ..coupling products":

It was very surprising, that the modified second compound can not only bind the target itself, but also act as a site-directing probe. By repeating the method with the modified second compound of the invention instead of the electrophilic used in the first round (a-d), the hits are improved due to the iteration.

Preferably the method can be used to transform a moderately active peptide inhibitor (electrophilic binder) to an entirely non-peptidic inhibitor with low μM inhibition. It is understood, that the iteration step of the method described above can be repeated several times.

Also preferred is the method, wherein the method further comprises a fusion of the modified second compound or a derivative thereof with the identified first compound and/or the identified third compound and/or a derivative thereof.

Surprisingly the identified and/or modified compounds can be coupled together to an extremely specific inhibitor for target.

Another preferred embodiment of the invention relates to the method, wherein the method further comprises a production of the second binding-species and/or derivatives thereof, wherein the modified second compound and/or a derivative thereof is ligated to the identified first compound and/or the identified third compound and/or a derivative thereof.

Another preferred embodiment is the method according one of the preceding claims, wherein the electrophile and/or the modified second compound and/or a derivative thereof are used as an active site-directing probe.

If one formation partner is known to bind to a defined site of the target this methodology allows the site-directed identification of binding fragments. Therefore non-specific binding is not an issue in the method of the invention. It was especially surprising that the modified second compound of the invention can be useful as a site-directing probe.

Also preferred is the method, wherein the first and/or the third compound are low-affinity binder, binding to the target with K_D > 100μM.

Binding affinities in the higher millimolar range cannot be detected in conventional high- throughput assays and in NMR screening the detection limit of lower affinities is defined by solubility of the tested small molecules. In method of the invention, however, surprisingly relative changes in affinity are observed. Considering the thermodynamics of protein- compound binding (see Formula A), a free binding energy of -1 kJ/mol for a compound- protein complex results in a dissociation constant of the complex (K₀) of 0.67 M (at 298 K). If however the same compound is ligated to a known inhibitor yielding a product with a free binding energy decreased by 1 kJ/mol, this will result in a reduction of the K_Dfor the ligation product of by more than 30 % (0.67^*K_D) which, in principle, can be detected by most standard assays. The method of the invention exploits this effect. Thereby it should enable the detection of low-affinity ligands by forming a dynamic ligation product with a site-directing inhibitor and measuring its activity in a classical biochemical assay.

ΔG AG, +ΔG, &G, ΔG,

Jζ - _p RT _ p RT _ _p RT * „ RT - Jζ * Jζ

Formula A.

A person skilled in the art knows the meaning of the parameters.

Also preferred is the method, wherein the detection assay is a enzyme assay and/or a protein assay, preferred wherein the binding of the first binding-species and/or the second binding-species and/or derivatives thereof to the target is determined via an amount of product formation in the enzyme assay and/or an fluorescence signal, fluorescence polarization signal, luminescence signal, UV/VIS absorption and/or radioactive signal in a protein assay preferred an ELISA or fluorescence polarization assay.

Surprisingly using an enzymatic reaction for compound detection reduces the required amount of target strongly.

Preferred is an enzymatic detection via the release of a fluorescent reporter molecule; surprisingly this setup enables high-throughput screening in microtiter plates, which has not been reported so far.

Another preferred embodiment relates to the method, wherein a substrate of the target is added, preferred a labeled substrate, preferred with a reporter molecule and wherein the substrate and the first binding-species or the second binding-species cannot bind to the target at the same time, preferred wherein they compete for the same active site of the target.

Also preferred is the method, wherein the electrophile binder is a substrate of the target, preferred a labeled substrate. For example 4-Formyl-phenyl-phosphate can act as a substrate and an electrophilic binder for protein tyrosine phosphatases. This embodiment is advantageous because no additional substrate needs to be added. Also preferred is the method, wherein the target is a macromolecule, selected from the group comprising a protein, an enzyme, a biological receptor, an antibody, a ribonucleic acid molecule and/or a deoxyribonucleic acid molecule.

Proteins and/or enzymes often play an important role in the pathogenesis of various diseases. Therefore they are an attractive target for this method. There are lots of enzymes, for which no binders especially inhibitors are available. In addition enzymes are an advantageous target, because one can use the enzymatic function in the parallel detection assay.

Biological receptors are also known to be involved in disease, thus there is a need for the identification of binders which can alter the receptor function. For example biological receptors can be over expressed in certain diseases, which requires the inhibition of the receptor. On the other hand, pathogens can have different receptors compared to mammals; therefore these receptors are a favorable target in the treatment of disease caused by these pathogens.

The identification of fragments which can bind antibodies is a very important issue in different fields. For instance the inhibition of antibodies can be used in the therapy of autoimmune disorders. Antibodies are also used in many detection assays, so that identified fragments which can bind to an antibody and e.g. alter its activity could be applied in different assays, used in laboratory routines.

The interaction with the regulation on DNA or RNA level to e.g. alter protein expression gets more and more important. In this context the identification of more binding fragments is necessary. For example DNA or RNA binder can block or enhance the synthesis of a special protein.

Another preferred embodiment relates to the method, wherein the enzyme is selected from the group comprising SARS coronavirus main protease, phosphotyrosine phosphates, preferred ptp1 B, MptpA, SHP-2, ptpN7, ptpRR, phosphotyrosine binding proteins, preferred STAT-5, human immunodeficiency virus protease-1 and/or β-amyloid converting enzyme-1.

SARS-CoV M^pro is a cysteine protease that is essential for replication of the coronavirus in- side the infected host cell. Thus, it has been identified as a drug target for the severe acute respiratory syndrome and potentially other coronavirus-caused diseases. Several irreversible (covalent) peptide-based inhibitors have been prepared and co-crystallized with the enzyme in the state of art. Peptide-based inhibitors are not well-suited for pharmaceutical compositions and irreversible inhibitors often induce side-effects. In the absence of effective drugs or a vaccine for SARS, control of this disease still relies on the rapid diagnosis and the appropriate management, including the isolation of suspect and probable cases and the management of their close contacts. The symptoms are similar to symptoms of influenza, which is why it is often difficult to diagnose the disease fast enough. Therefore it is a need to identify new inhibitors and to create a pharmaceutical composition affective to SARS.

Phosphotyrosine phosphates play an important role in various signal transduction pathways. The discovery of fragments which can bind to phosphotyrosine phosphates and by that for example regulate, inhibit, activate or label these enzymes could be used in research as well as in the therapy of disorders.

PTP1 B is a protein tyrosine phosphatase and serves as a key negative regulator of the tyrosine phosphorylation cascade integral to the insulin signaling pathway. PTP1 B is also known to be up regulated in patients with breast cancer and some other cancer disease too. Therefore it is obvious that there is a need for compounds that interact with PTP1 B.

Protein tyrosine phosphatases from several microorganisms have been shown to modify the phosphorylation/dephosphorylation equilibrium in their host cells and by that act as virulence factors. MptpA is secreted by Mycobacterium tuberculosis, a bacterium which is wide spread and often resistant to antibiotics. Thus the identification of binding fragments would be beneficial.

SHP-2 is a non-receptor-phosphotyrosine-phosphorylase that possesses a central regulatory function in many signal transduction pathways in particular signal transduction for growth factors. Therefore inhibitors are needed for the treatment of various diseases but also for research for example the analyses of signal pathways.

Protein tyrosine phosphatase non-receptor type 7(ptpN7) and protein tyrosine phosphatase receptor type R (ptpRR) both have the ability to specifically inactivate MAPKs (mitogen- activated protein kinases). Binder fragments could be important e.g. for treating neurodegenerative diseases in the case of ptpRR or acute myeloblasts leukaemia targeting ptpN7. The Signal Transducers and Activators of Transcription (STAT) are involved in various pathways. Defects of STAT can result in serious diseases. Thus STAT-inhibitors could be used in treatments and in research as well.

HIV is one of the most serious health problems worldwide. So far there is no therapy avail- able to cure AIDS; therefore it is essential to identify more compounds which could possibly alter the function of viral enzymes like HIV-protease-1.

Another preferred target is BACE-1 , a key protein in the pathogenesis of Alzheimer's disease. Obviously finding a cure for Alzheimer's disease is of major importance. So far no sat- isfying treatment is available, which is why the identification of new binding compounds is necessary. It is understood that these are just examples and preferred embodiments of the invention. The skilled artisan has the knowledge to adapt the disclosed method of the invention to any target of interest without being inventive himself.

Another preferred embodiment relates to the method, wherein the electrophilic binder is selected from the group comprising Ac-DSFDQ-H (SEQ ID No. 1 : DSFDQ), carboxyfluo- resceine DEVD-CO-CHO (SEQ ID No. 2: DEVD), Ac-DEVD-CO-CHO, 4-formyl-phenyl- phosphate, 4-formylphenyl dihydrogen phosphate, the compound of formula 2, the compound of formula 3 and/or the compound of formula 4.

Formula 2:

Formula 3:

Also preferred is the method, wherein the detection assay comprises an enzymatic detection via a release of a reporter molecule, preferred a fluorescent reporter molecule and/or a protein binding assay performed by fluorescence, fluorescence polarization, luminescence, UV/Vis absorption or radioactive signal like fluorescence polarization assay or ELISA.

Various assays can be introduced into the method. Preferred is the method using a "substrate competition assay" as a detection assay. According to the invention a "substrate competition assay" works as follows: A substrate, preferred a labeled substrate, of the target is used. When one of the binding- species binds to the target, the substrate cannot be converted by the target anymore. One possibility is that substrate and binding-species (e.g. the electrophilic binder, the nucleophile or the ligation product thereof) compete for the same site of the target. It is also possible that bind to different sites of the target but that binding of the binding-species prevents binding of the substrate, for example via changes in the conformation. Any mechanism that leads to less binding of the substrate to the target, due to binding of the binding-species can take place in the "substrate competition assay" of the invention.

It is preferred that less binding of a substrate leads to less substrate conversion. It is further preferred, that the change in converted substrate is detected via the assay. For example it is possible that due to the conversion of substrate a reporter molecule is released. In this case the binding of a binding-species is indicated by a rate of release. This assay is especially preferred for the targets SARS-CoV M^pr0, HIV protease and/or BACE-1.

Another preferred assay used as a detection assay in the method of the invention is an assay with "substrate amplification". In this set up it is preferred that the electrophilic binder itself is a substrate of the target. This assay is advantageous because no additional substrate is needed. The binding of a compound and/or a binding-species to the target is indicated by a higher substrate conversion. This assay is also advantageous because it is possible to identify competitive binding of a compound. This means that binding-species and compounds identified via this method compete for the same site as the substrate. Surprisingly this assay showed extremely good results in the method with a protein tyrosine phosphatase as a target. It was especially useful for the detection of protein-specific fragments.

Using an enzymatic reaction for fragment detection reduces the required amount of protein strongly. An enzymatic detection via the release of a fluorescent reporter molecule enables high-throughput screening in microtiter plates.

Also preferred are detection assays that indicate the binding via an altered fluorescent polarization. One alternative is the increase of a fluorescent polarization (fluorescence polariza- tion amplification or binding amplification assay). This assay showed very promising results, especially for STAT5 as a target. Especially preferred is the use of the compounds of formula 3 and/or 4 in this assay.

Also preferred is a detection assay working via "fluorescent polarization competition". This kind of assay can be used advantageous for STAT5 as a target. A substrate labeled with a reporter molecule is used. The target is converting the substrate which leads to the release of the reporter molecule. Therefore the binding of a compound and/or a binding-species is indicated via a decreased release of reporter molecule and therefore a decreased fluorescent polarization.

Also preferred is the method, wherein the reporter molecule is 7-amino-4-methyl-3- coumarinylacetic amide and/or a carboxyfluoresceine labelled binding molecule preferred carboxyfluoresceine-DEVD-CO-CHO or an absorbing or fluorescent heavy metal phosphate complex.

Also preferred is the method, wherein the detection assay is performed as a high-throughput assay, preferred in microtiter plates.

Another preferred embodiment of the invention relates to the method, wherein the method is performed as a high-throughput method, preferred in microtiter plates. The method of the invention is superior, because no additional equipment is required besides a standard microtiter plate reader. Therefore no additional costs will be incurred. Preferably the method is carried out in a single reaction vessel, which simplifies the realisation of the high-throughput method.

One major advantage of the invention is the fact that the detection assay is performed at the same^ time as the compounds are identified. Therefore the high-throughput method comprises the detection assay.

A high throughput screening provides the ability to test large numbers of compounds quickly and efficiently. Especially the use of microtiter plates is advantageous due to minor costs. Because only few components need to be combined in a small reaction volume, the automated pipetting of the reaction and execution in 96-well or 384-well microtiter plates can be easily performed using standard laboratory robots. Each well can contain a different compound that is combined with a mastermix containing the remaining reaction components. Preferred the detection assay can also be executed in the same process on a laboratory robot, which allows the processing large numbers of samples. This is a great advantage compared to the screening methods in the prior art.

Also preferred is the method, wherein the second binding-species comprises a molecule of formula 1 :

(1)

The compound of formula (1) is a surprisingly strong inhibitor for SARS-CoV M^pro. This com- pound is superior to all inhibitors for SARS-CoV M^pra known in the state of art. The compound of formula (1) is characterized by a high specificity and efficacy. Surprisingly the compound is well-suited for the use in a pharmaceutical composition.

Also preferred is the method, further comprising formulating the first compound, the second compound, the third compound, the first binding-species and/or the second binding-species and/or derivatives thereof in a pharmaceutically acceptable form.

Another preferred embodiment of the invention relates to the method for the production of a pharmaceutical composition comprising the method of at least one of the preceding claims and furthermore mixing the first compound, the second compound, the third compound, the first binding-species and/or the second binding-species and/or derivatives thereof with a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable" is employed herein to refer to those com- pounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Also preferred is the method, wherein the carrier is selected from the group comprising fillers, disintegrants, binders, humectants, extenders, dissolution retarders, absorption enhancers, wetting agents, adsorbents and/or lubricants.

The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as so- dium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene gly- col; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavouring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

The invention also relates to the first compound, the second compound, the third compound, the first binding-species, the second binding-species and/or derivatives thereof produced by the method.

Preferred is the second binding-species, especially preferred according to formula I, the second compound, the first binding-species and/or derivatives thereof produced by the method.

The method of the invention can not only be used to identify low-affinity binders, but also to produce compounds and binding-species.

Surprisingly the synthetic combination of hit fragments (the compounds and/or binding- species of the invention) detected in the two rounds of optimization yielded in an inhibitor with increased activity. The inhibitors of the invention are highly specific and therefore suitable for pharmaceutical approaches. Preferred is a reversible inhibitor because less side- effects are monitored. However, for some targets irreversible inhibitors are preferred, depending on the assigned problem. A person skilled in the art is well aware of that and therefore knows when an irreversible inhibitor is suited.

The invention also relates to a pharmaceutical agent comprising the first compound, the second compound, the third compound, the first binding-species, the second binding-species and/or derivatives thereof and a pharmaceutically acceptable carrier.

Preferred is the pharmaceutical agent according to the preceding claim, characterized in that said agent is a capsule, a tablet, a coated tablet, a suppository, an ointment, a cream, an injection solution, an infusion solution, a vaginal and/or rectal suppository, a pad and/or a foam.

In a preferred embodiment, (i) the fragments and the electrophilic binder as an active site directing probe are in a very low concentrated dynamic equilibrium and (ii) the binding activity of the reversibly formed ligation product of the fragment and the electrophilic aldehyde binder is indicated by the determination of an altered amount of product formation in an enzyme assay or the stronger or weaker fluorescence, fluorescence polarization, luminescence, UV/VIS absorption or radioactive signal in a protein assay e.g. enzyme linked immuno assay (ELISA) or fluorescence polarization assay.

In this embodiment the electrophilic binder acts as an active site directing probe. By this feature the electrophilic binder can direct the fragment to an active site of the target. This enables the identification of low-affinity fragments, which are not able to bind the target by themselves. Only very low concentrations of fragments and electrophilic binders are necessary for the method of the invention, which leads to fewer costs and the prevention of unspecific binding.

The teachings of the present invention are characterised by the following features:

- departure from the beaten track a new perception of the problem satisfaction of a long-felt need or want hitherto all efforts of experts were in vain the simplicity of the solution, which proves inventive action, especially since it re- places a more complex doctrine the development of scientific technology followed another direction the achievement forwards the development misconceptions among experts about the solution of the according problem (prejudice) - technical progress, such as: improvement, increased performance, price-reduction, saving of time, material, work steps, costs or resources that are difficult to obtain, improved reliability, remedy of defects, improved quality, no maintenance, increased efficiency, better yield, augmentation of technical possibilities, provision of another product, opening of a second way, opening of a new field, first solution for a task, spare product, alternatives, possibility of rationalisation, automation or miniaturisation or enrichment of the pharmaceutical fund special choice; since a certain possibility, the result of which was unforeseeable, was chosen among a great number of possibilities, it is a patentable lucky choice error in citations - young field of technology combined invention; a combination of a number of known elements, with a surprising effect licensing praise of experts and - commercial success

Said advantages are shown especially in the preferential embodiments of the invention. The examples described in the following demonstrate how the invention can be carried out. It is understood, that these are just examples and that the invention is nit limited to these embodiments.

Examples

Example 1 : The concept of the method of the invention in a preferred embodiment (figure

1) The electrophile acts as a directing probe (circle) for the active site of the protein in competition with the AMCA-labeled substrate. Cleavage of the substrate leads to release of the AMCA fluorophore and thus to increased fluorescence. The electrophile reacts in equilibrium with the nucleophilic fragment (square), directing it into the proximate subpocket. Consequently, the increase of AMCA fluorescence detected depends on the affinity of the formed imine to the protein. The electrophile and also the formed imine are expected to react re- versibly with the active-site cysteine.

It is understood, that this example is just an illustration and that the invention is not limited to this exact method.

The following examples demonstrate the synthesis for certain substrates and electrophiles. These are selected examples to demonstrate how the invention can be carried out. It is understood that every other possibility, described in the application, is also part of the invention, therefore the scope of the invention shall not be limited to these examples.

Example 2: Synthesis of fluorogenic substrate Ac-TSVALQ-AM CA

7-Fmoc-amino-4-methyl-3-coumarinylacetic acid (Fmoc-AMCA) was prepared as described in literature (Harris JL et al. 2000, Proc Natl Acad Sci).

Yield: 930 mg, 95%. ¹H-NMR: (300 MHz): δ = 2.33 (s, 3 H₁ CH₃), 3.51 (m, 2 H, CH₂), 4.34 (m, 1 H, Fmoc CH), 4.52 (m, 2 H, Fmoc CH₂), 7.33- 7.44 (m, 4 H, Fmoc Ar CH), 7.56 (s, 1 H, AMCA Ar CH), 7.62 (m, 2 H, AMCA Ar CH), 7.78- 7.85 (m, 4 H, Fmoc Ar CH), 10.19 (s, 1 H, CONH) ppm. ¹³C-NMR: (300 MHz): δ = 15.16 (AMCA CH₃), 33.28 (AMCA CH₂), 46.80 (Fmoc CH), 66.21 (Fmoc CH₂), 106.58 (AMCA Ar CH) 116.17 (AMCA Ar CH), 117.68 (AMCA Ar quat C), 119.93 (Fmoc Ar CH), 122.06 (AMCA Ar quat C), 124.85 (Fmoc Ar CH), 127.28 (Fmoc Ar CH), 127,70 (Fmoc Ar CH), 128.30 (AMCA Ar CH), 141.06 (AMCA CNH), 142.36 (Fmoc quat C), 143.90 (Fmoc quat C), 145.47 (AMCA Ar quat CCH₃), 153.53 (Fmoc CO), 161.14 (AMCA Ar CO), 171.81 (AMCA COO) ppm.

Fmoc-AMCA was immobilized on Rink Amide resin by DIC/HOBt method in DMF. Fmoc- AMCA (956.5 mg, 2.1 mmol), HOBt (284.6 mg, 2.1 mmol) and DIC (336 μL, 2.1 mmol) were solved in DMF (4 mL) and were given to the Fmoc-deprotected Rink Amide resin (1 g, 0.7 mmol/g). The efficient substitution of the resin was totally with 0.7 mmol/g as determined by spectrophotometric Fmoc quantification.

The Fmoc group was cleaved by 20% piperidine in DMF (6 min) from the Fmoc-AMCA-resin (1 g, 0.7 mmol/g). FmocGln(Trt)-OH (1710 mg, 2.8 mmol) was dissolved in DMF (4 mL) by addition of collidine (742 μL, 5.6 mmol), HATU (1064 mg, 2.8 mmol). Then the mixture was added to the resin (1 g, 0.7 mmol/g), followed by agitation overnight. Spectrophotometric Fmoc-quantification assay showed totally coupled amino acid to the AMCA-resin (0.7 mmol/g). Synthesis of the peptide sequence TSAVL was carried out manually, according to the DIC/HOBt method described above. After final Fmoc-deprotection, the N-terminus was ac- etylated by treatment with Ac₂O (5 eq in 4 mL DMF) for 20 min. Cleavage of the side-chain protecting groups and of the resin was performed in a single step by treatment with a mixture of trifluoroacetic acid/water/triisopropylsilane (95:2.5:2.5, v:v:v) for three hours at room temperature. The crude product was precipitated by adding cold diethylether and collected by centrifugation. The obtained fluorogenic substrate was lyophilized from water. The products were characterized using HPLC-ESI-MS.

Ac-TSAVLQ-AMCA (1): Yield: 374 mg, 61%. ESI/MS: calcd [M = C₄₀H₅₉N₉O₁₃], 873.0 Da; found (MH⁺), m/z 874.0 Da.

Example 3: Synthesis of Λ/-(3-AcDFSDQ(ψCH₂)-aminophenyl)-3-aminobenzamide (10) N-Fmoc-3-aminobenzoic acid was prepared analogous as described in literature (Harris JL et al 2000, Proc Natl Acad Sci). Yield: 5.07 g, 95%. ¹H-NMR: (300 MHz): δ = 4.29 [t, 1 H, Fmoc CH, J = 6.3 Hz], 4.46- 4.48 (d, 2 H, Fmoc CH₂, J = 6.7 Hz), 7.29- 7.45 (m, 4 H₁ Fmoc Ar CH), 7.54- 7.66 (m, 4 H, Fmoc Ar CH), 7.71- 7.89 (m, 3 H, Ar CH), 8.25 (s, 1 H₁ Ar CH), 9.98 (s, 1 H, NH) ppm. ¹³C-NMR: (300 MHz): δ = 46.44 (Fmoc CH)₁ 65.57 (Fmoc CH₂), 118.88 (Fmoc Ar CH), 120.03 (Ar CH), 122.37 (Ar CH), 123.24 (Ar CH), 125.06 (Fmoc Ar CH)₁ 127.01 (Fmoc Ar CH), 128.87 (Ar C(CO)), 131.19 (Ar CH), 139.53 (Ar C-NH), 141.40 (Fmoc C), 143.70 (Fmoc C), 153.54 (O(CO)NH), 166.70 ((CO)OH) ppm.

1 ,3-Diaminobenzene was immobilized on 2-chlorotrityl chloride resin: To a suspension of 1 ,3- diamino-benzene (1081 mg, 10 mmol) in DCM (2 mL), DIPEA (1 ,7 mL, 10 mmol) was added and followed by DMF until complete dissolution. The mixture was added to the 2-chlorotrityl resin (1 g) and shaken overnight. Unreacted chloro functions were quenched by treatment with methanol for 5 min. Loading was determined from the substitution of the Fmoc-Leu by Fmoc-specrometric assay. The load was 0.2 mmol/g. The amino group of immobilized m- diamino-phenyl (1 g, 0.2 mmol/g) was acylated by 3-Fmoc-aminobenzoic acid. The 3-Fmoc- aminobenzoic acid (2.88 g, 0.8 mmol), collidine (212 μL, 0.8 mmol), and HATU (304 mg, 0.8 mmol) were solved in DMF and added to the resin, followed by agitation overnight. HPLC- ESI-MS showed totally coupled acid to the m-diaminophenyl.

The Fmoc-(3-amino-Λ/-(3-amino-phenyl)-benzamide 2-chlorotrityl resin was Fmoc depro- tected by 20% piperidine in DMF for 6 min. Fmoc-Gln(Trt) aldehyde (417 mg, 0.7 mmol) was solved in dry DMF containing 1% acetic acid (AcOH) and was added to the resin (1 g, 0.2 mmol/g). After 10 min the reducing agent NaCNBH₃ (132 mg, 2.1 mmol) was added to the reaction vessel. After 3 h the resin was washed. The successful reaction was determined by spectrophotometric Fmoc quantification.

Synthesis of the peptide sequence DSFD was carried out manually, according to the DIC/HOBt method as described above. After final Fmoc-deprotection, the N-terminus was acetylated by treatment with Ac₂O (5 eq in 4 mL DMF) for 20 min. Cleavage of the side-chain protecting groups and of the resin was performed in a single step by treatment with a mixture of trifluoroacetic acid/water/triisopropylsilane (95:2.5:2.5, v:v:v) for three hours at room tern- perature. The crude product was precipitated by adding cold diethylether and collected by centrifugation. The obtained product was lyophilized from water. The product was characterized using HPLC-ESI-MS.

Yield: 95 mg, 55%. ESI/MS: calcd [M = C₄₀H₄₇N₉O₁₃], 861.0 Da; found (MH⁺), m/z 862.0 Da.

Example 4: Synthesis of Ac-DSFDQ-(ψCH₂-NH₂) (11)

The Sieber Amide resin (100 mg, 0.71 mmol/g) was Fmoc-deprotected by 20% piperidine in DMF. Fmoc-Gln-(Trt) aldehyde (417 mg, 0.7 mmol) was solved in dry DMF containing 1% acetic acid (AcOH) and was added to the resin. After 10 min the reducing agent NaCNBH₃ (132 mg, 2.1 mmol) was added to the reaction vessel. After 3 h the resin was washed. The successful reaction was determined by spectrophotometric Fmoc quantification. Synthesis of the peptide sequence DSFD was carried out manually, according to the DIC/HOBt method as described above. After final Fmoc-deprotection, the N-terminus was acetylated by treatment with Ac₂O (5 eq in 4 mL DMF) for 20 min. Cleavage of the side-chain protecting groups and of the resin was performed in a single step by treatment with a mixture of trifluoroacetic acid/water/triisopropylsilane (95:2.5:2.5, v:v:v) for three hours at room temperature. The crude product was precipitated by adding cold diethylether and collected by centrifugation. The obtained product was lyophilized from water. The product was characterized using HPLC-ESI-MS. Ac-DSFDQ-(ψCH₂-NH₂) (11): Yield: 37 mg, 79%. ESI/MS: calcd [M = C₂₇H₃₉N₇O₁₁], 637.0 Da; found (MH⁺), m/z 638.0 Da.

Example 5: Synthesis of peptides DSFDQ-OH, Ac-DSFDQ-OH, and AcDSFDQ-NH₂ Synthesis of the peptide sequence DSFD was carried out manually, according to the DIC/HOBt method described above, on Rink Amide resin and 2-chlorotrityl chloride resin.

Ac-DSFDQ-OH (12): ESI/MS: calcd [M = C₂₇H₃₆N₆O₁₃], 652.0 Da; found (MH⁺), m/z 653.0

Da. DSFDQ-OH (13): ESI/MS: calcd [M = C₂₅H₃₄N₆O₁₂], 610.0 Da; found (MH⁺), m/z 611.0

Da.

Ac-DSFDQ-NH₂ (14): ESI/MS: calcd [M = C₂₇H₃₇N₇O₁₂], 651.0 Da; found (MH⁺), m/z 652.0 Da.

Example 6: Synthesis of 3-acetylamino-Λ/-[3-(2-oxo-ethyl)-phenyl-benzamide (15) 3-Acetylamino-benzoic acid (197 mg, 1.1 mmol) and EDC (170 mg, 1.1 mmol) were suspended in DCM (6 mL) and stirred for 10 min. Then, 2-(3-aminophenyl)-ethanol (151 mg, 1.1 mmol) was added and stirred overnight. The solution was diluted with AcOEt (30 mL) and washed with 0.1 N HCI (3 x 25 mL). Afterwards, the organic phase was washed with saturated Na₂CO₃ solution (3 x 25 mL) and dried over MgSO₄. 3-Acetylamino-N-[3-(2- hydroxyethyl)phenyl]-benzamide (0.16 mmol, 48 mg) was obtained by evaporation and dissolved in 5 mL DCM. Dess-Martin-periodinane (5 eq., 0.8 mmol, 339 mg) was added, fol- lowed by 500 μL of water. The mixture was stirred until oxidation was complete, as checked by TLC. The work-up was performed by adding 30 mL AcOEt. The organic phase was washed twice with saturated NaHCO₃ solution, H₂O, and brine, dried with MgSO₄, filtered and evaporated to dryness. Finally, a yield of 8.9 mg, 19% of the desired product was obtained. ESI/MS: calcd [M = C₁₇H₁₆N₂O₃], 296.1 Da; found (MH⁺), m/z 297.0 Da. ¹H-NMR: (300 MHz): δ = 2.07 (s, CH₃), 3.74 (s, CH₂), 6.96-6.99 (d, Ar CH, J= 7.2 Hz), 7.29- 7.32 (dd, Ar CH, J= 2.7 Hz, J'= 2.7 Hz), 7.40- 7.45 (dd, Ar CH, J= 7.8 Hz, J'= 7.8 Hz), 7.58- 7.61 (d, Ar CH, J= 7.5 Hz), 7.65-7.67 (m, Ar CH), 7.79-7.81 (d, Ar CH, J= 7.5 Hz), 8.05 (s, Ar CH), 9.69, 10.11 , 10.16, 10.23 ppm. ¹³C-NMR: (300 MHz): δ = 23.95 (CH₃), 49.66 (CH₂), 118.48, 118.91 , 121.43, 121.87, 125.02, 128.64, 128.94, 133.10, 135.61 , 139.41 , 165.55 (CONH₂), 168.48 (CONH₂), 200.27 (CHO) ppm.

Example 7: Synthesis of 3-acetylamino-Λ/-(3-formyl-phenyl)-benzamide (16)

3-acetylamino-benzoic acid (197 mg, 1.1 mmol) and EDC (170 mg, 1.1 mmol) were suspended in DCM (6 mL) and stirred for 10 min. Then 3-aminobenzylalcohol (135 mg, 1.1 mmol) was added and stirred overnight. Next, 30 mL of AcOEt were added and the solution was washed with 0.1 N HCI (3 x 25 mL) to eliminate the EDC and some starting material with a free amino group. Afterwards, the organic phase was washed with saturated Na₂CO₃ solution (3 x 25 mL) to eliminate products with a free carboxylic function.

0.17 mmol (48 mg) of 3-acetylamino-Λ/-[3-(2-hydroxyethyl)phenyl]-benzamide were obtained and dissolved in 5 mL DCM. Then, 5 eq. of Dess-Martin-periodinane (0.9 mmol, 382 mg) were added, followed by 500 μL water. The mixture was stirred until full oxidation had been reached, as checked by TLC. The work-up was performed by adding 30 mL AcOEt. The organic phase was washed twice with saturated NaHCO₃ solution, H₂O, and brine, dried with MgSO₄, filtered and evaporated to dryness. Finally, we obtained a yield of 24.8 mg, 52% of the desired product were obtained. ESI/MS: calcd [M = C₁₆H₁₄N₂O₃], 282.1 Da; found (MH⁺), m/z 283.0 Da. ¹H-NMR: (300 MHz): δ = 2.85 (s, 3 H, CH₃), 7.42- 7.48 (dd, 1 H, Ar CH, J = 8.0 Hz, J'= 8.0 Hz), 7.56- 7.67 (m, 3 H, Ar CH), 7.80- 7.83 (d, 1 H, Ar CH, J = 8.1 Hz), 8.03- 8.06 (d, 1 H, Ar CH, J = 8.1 Hz), 8.10 (s, 1 H, Ar CH), 8.36 (s, 1 H, Ar CH), 10.00 (s, 1 H, NH), 10.13 (s, 1 H, NH), 10.50 (s, 1 H, CH), ppm. ¹³C-NMR: (300 MHz): δ = 23.95 (CH₃), 118.51 , 120.08, 121.94, 122.14, 125.34, 126.02, 128.73, 129.53, 135.24, 136.66, 139.49, 139.94, 165.83 (CONH₂), 168.52 (CONH₂), 193.01 (CHO).

Example 8: Synthesis of 3-acetylamino-N-[3-(2,3-dioxo-propyl)-phenyl]-benzamide (17) and 3-Acetylamino-N-[3-(2-oxo-acetyl)-phenyl]-benzamide (18)

Triphenylphosphine resin (2 g, 1.6 mmol/g, 3.2 mmol, 1 % divinyl benzene, 100-200 mesh) was weighed into a microwave vial and suspended in dry toluene (16 mL). After addition of teff-butyl 2-bromoacetate (2.36 mL, 16 mmol, 5 eq), the vial was shaken for 24 h at room temperature. Then, the vial was washed with dry toluene and DCM. The obtained polymeric phosphonium salt was suspended in dry DCM (4 mL) and TEA (2.23 mL, 16 mmol, 5 eq) was added. After shaking for 2 h at room temperature, the resin was filtered, washed, and dried in vacuo.

Example 9: 3-Acetylamino-N-[3-(2,3-dioxo-propyl)-phenyl]-benzamide (17) A portion of 700 mg of the tert. -butyl phosphoranylidene acetate resin was swollen in DCM in 25 mL polyethylene syringes equipped with a polypropylene filter. After washings with DCM, acylation of the resins was carried out using Λ/-Fmoc-3-aminophenylacetic acid (2.27 g, 5.6 mmol), BTFFH (1.77g, 5.6 mmol), and DIPEA (1.95 mL, 11.2 mmol) in 8.5 mL of dry DMF for 20 h. Then, the resin was washed with DMF and DCM and dried in vacuo. Loadings were determined by Fmoc UV titration of small aliquots of the resins (Λ/-Fmoc-3- aminophenylacetic acid = 1.37 mmol/g). Fmoc group was removed in the usual way and the resulting free amino group acylated shaking the resin with 3-acetylaminobenzoic acid (1 g, 5.6 mmol), BTFFH (1.77 g, 5.6 mmol), and DIPEA (1.95 mL, 11.2 mmol) in 6 mL of dry DMF for 6h. After that, washings with DMF and DCM were carried out.

Terf-butyl removal: the resin was treated with 6 mL of TFA:DCM (95:5) for 18 h (the standard 1h treatment for the removal of terf-butyl group on solid phase gave only 20 % removal). After washing the resin with DCM (5x), neutralization was carried out by treating the resin with DIPEA:DCM (1 :9, v:v) (3 x 5 min) followed by more DCM washings. Cleavage of the com- pound from the resin was carried out by treating 350 mg of the resin solvated in the minimum dry DCM with 5 eq of DMDO (507.5 mg, 6.6 mmol) for 1h at O⁰C. The resin was filtered and the filtrate was evaporated to dryness, suspended in H₂O:AcCN (1 :1 , v:v), frozen and lyophi- lized. 96 mg (75 % purity) of the target product as a yellow solid were obtained. The product was purified by semipreparative HPLC and characterized by LC-MS and NMR. ESI/MS: calcd [M = C₁₈H₁₆N₂O₆], 324.34 Da; found (MH⁺), m/z 325.0 Da. ¹H-NMR: (300 MHz): δ = 2.17 (s, CH₃), 3.74 (s, CH₂), 6.96-6.99 (d, Ar CH, J= 7.2 Hz), 7.29- 7.32 (dd, Ar CH, J= 2.7 Hz, J'= 2.7 Hz), 7.40- 7.45 (dd, Ar CH, J= 7.8 Hz, J'= 7.8 Hz), 7.58- 7.61 (d, Ar CH, J= 7.5 Hz), 7.65-7.67 (m, Ar CH), 7.79-7.81 (d, Ar CH, J= 7.5 Hz), 8.05 (s, Ar CH), 9.69, 10.11 , 10.16, 10.23 ppm. ¹³C-NMR: (300 MHz): δ = 23.49 OCH₃), 48.61 (CH₂), 117.08, 120.35, 120.38, 124.36, 125.40, 125.44, 128.41 , 130.16, 133.81 , 134.71 , 137.68, 138.85, 164.10 (CO), 168.29 (NHCO), 189.89 (CHO), 190.86 (CHOCO) ppm.

Example 10: 3-Acetylamino-N-[3-(2-oxo-acetyl)-phenyl]-benzamide (18)

A portion of 700 mg of 2-phosphoranylidene acetate resin was swollen in DCM in 25 mL polyethylene syringes equipped with a polypropylene filter. After washings with DCM, acylation of the resins was carried out using Λ/-Fmoc-3-aminobenzoic acid (2.01 g, 5.6 mmol), BTFFH (1.77g, 5.6 mmol) and DIPEA (1.95 mL, 11.2 mmol) in 8.5 mL of dry DMF for 2Oh. Then, the resins were washed with DMF and DCM and dried in vacuo. Loadings were de^¬ termined by Fmoc UV titration of small aliquots of the resin (Λ/-Fmoc-3-aminobenzoic acid = 1.15 mmol/g). The Fmoc group was removed in the usual way and the resulting free amino group acylated shaking the resin with 3-acetylaminobenzoic acid (1 g, 5.6 mmol), BTFFH (1.77 g, 5.6 mmol), and DIPEA (1.95 mL, 11.2 mmol) in 6 mL of dry DMF for 6h. After that, washings with DMF and DCM were carried out. Te/f-butyl removal: the resin was treated with 6 mL of TFA:DCM (95:5) for 18 h (the standard 1h treatment for the removal of tert-butyl group on solid phase gave only 20 % removal). After washing the resin with DCM (5x), neutralization was carried out by treating the resin with DIPEA:DCM (1 :9, v:v) (3 x 5 min), followed by more DCM washings. Cleavage of the com- pound from the resin was carried out by treating 700 mg of the resin solvated in the minimum dry DCM with 3 eq of DMDO (255.6 mg, 3.45 mmol) for 45 min at O⁰C. The resin was filtered and the filtrate was evaporated to dryness, suspended in H₂OiAcCN, frozen and lyophilized. After lyophilization, the resulting yellow solid was suspended in 10 ml_ of H₂O:AcCN (1 :1, v:v) and filtered to get rid of insoluble impurities. After evaporating the filtrate to dryness, 80.2 mg (80 % purity) of the target product were obtained as a yellow solid. The product was purified by semipreparative HPLC and characterized by LC-MS and NMR. ESI/MS: calcd [M = C₁₇H₁₄N₂O₆], 310.31 Da; found (MH⁺), m/z 311.0 Da. ¹H-NMR: (300 MHz): δ = 2.20 (s, 3 H, CH₃), 7.42- 7.48 (dd, 1 H, Ar CH, J = 8.0 Hz, J'= 8.0 Hz), 7.56- 7.67 (m, 3 H, Ar CH), 7.80- 7.83 (d, 1 H, Ar CH, J = 8.1 Hz), 8.03- 8.06 (d, 1 H, Ar CH, J = 8.1 Hz)₁ 8.10 (s, 1 H, Ar CH), 8.36 (s, 1 H₁ Ar CH), 9.04 (s, 2 H, NH), 9.64 (s, 1 H, CH)₁ ppm. ¹³C-NMR: (300 MHz): δ = 23.49 OCH₃), 117.08, 120.35, 120.38, 124.36, 125.40, 125.44, 128.41 , 130.16, 133.81 , 134.71 , 137.68, 138.85, 164.10 (CO), 168.29 (NHCO), 189.89 (CHO), 190.86 (CHOCO) ppm.

Example 11 : Synthesis of 1-(4-Amino-furazan-3-yl)-5-methyl-1H-[1 ,2,3]triazole-4-carboxylic acid ethyl ester (19)

The synthesis was performed accorded to a published procedure (Tselinski V, Mel'nikova SF, Vergizov SN, 1981 , Organicheskoi Khimii). 3-Amino-4-azidofurazan was obtained in a yield of 212 mg, 63 %. Observed melting point: 86 ⁰C (literature (Tselinski V, Mel'nikova SF, Vergizov SN, 1981 , Organicheskoi Khimii): mp: 86.5-87.5 ⁰C).

1-(4-Amino-furazan-3-yl)-5-methyl-1 H-[1,2,3]triazole-4-carboxylic acid ethyl ester was obtained in a yield of 60 mg, 75 %. Observed melting point: 138 ⁰C (literature: mp: 138-139 ⁰C).

Example 12: Reductive amination

As described in the literature (Groarke et al. 2000, Bioorg Med Chem Let.) compound 18 (13 mg, 38.9 μmol) and 1-(4-Amino-furazan-3-yl)-5-methyl-1H-[1 ,2,3]triazole-4-carboxylic acid ethyl ester 19 (9.3 mg, 38.9 μmol) were stirred in dry DMF/MeOH (1 :1 , v:v) containing 1% AcOH and molecular sieves for 3 h. Then 5 eq. Of CI₃SiH were added and stirred for 1 h. The molecular sieves were filtered off the filtrate was evaporated in vacuo to dryness. The crude product was purified by preparative HPLC. Finally a yield of 6 mg, 29 % was obtained. 22 ESI/MS: calcd [M = C₂₅H₂₄N₈O₆], 532.53 Da; found (MH⁺), m/z 533.0 Da. ¹H-NMR: (300 MHz): δ = 1.48 (t, CH2CH₃ J = 5.8 Hz), 2.20 (s, COCH3), 2.72 (s, CH₃), 3.74 (s, CH2), 4.56 (q, CH₂, J= 7.1 Hz), 6.96-6.99 (d, Ar CH₁ J= 7.2 Hz)₁ 7.29- 7.32 (dd, Ar CH₁ J= 2.7 Hz, J- 2.7 Hz), 7.40- 7.45 (dd, Ar CH, J= 7.8 Hz₁ J'= 7.8 Hz)₁ 7.46 (s, 3H₁ NH)₁ 7.58- 7.61 (d, Ar CH₁ J= 7.5 Hz), 7.65-7.67 (m, Ar CH), 7.79-7.81 (d, Ar CH, J= 7.5 Hz), 8.05 (s, Ar CH) ppm. ¹³C- NMR: (300 MHz): δ = 7.99 (ArCH₃), 13.87 (CH₃), 23.49 (COCH₃), 60.72 (OCH₂), 117.10, 120.98, 124.36, 125.40, 127.12, 129.48, 130.16, 134.67, 134.71 , 138.85, 141.53, 144.23, 162.93 (COO), 164.10 (CONH), 168.29 (COCH₃), 168.47 (NHCOCH₂) ppm.

Example 13: Kinetic analysis of fluorogenic substrate The activity of SARS-CoV M^pr0 was determined by measuring the release of AMCA. The excitation wavelength was set to 380 nm and the emission wavelength to 460 nm; relative fluorescence unit (RFU) λe_m 460 nm 63.861 RFU/μM AMCA. The sequence of the fluorogenic substrate used was Ac-TSAVLQ].AMCA. Cleavage reactions were incubated at 298 K and contained 1 μM SARS-CoV M^pro, 100 mM MES pH 7.0, and different concentrations of the fluorogenic substrate (0.25 mM - 2.5 mM) in a total volume of 20 μl. All measurements were carried out on a TECAN SAFIRE (Grailsheim, Germany). The K_M and V_max values for the cleavage of fluorogenic substrates by SARS-CoV M^pro were determined by measuring and plotting the initial rate, V, over a range of substrate concentrations S and fitting the data di- rectly into the Lineweaver-Burk-plot.

Example 14: Composition of a library of nucleophilic fragments

The subset of nucleophilic fragments used for the screening was derived from the rationally assorted fragment-based screening collection of the Leibniz Institute for Molecular Pharma- cology, Berlin. The 20,000 low-molecular-weight compounds of this library had been selected due to their diverse representation of reportedly bioactive scaffold elements and in compliance with physicochemical criteria, including the Lipinski rules.

In a first step, approximately 3,000 amines and thiols among the whole library of 20,000 compounds were identified as nucleophilic fragments. From this rationally composed frag- ment library containing 3000 nucleophilic fragments, a collection of 234 fragments was selected by a diversity analysis and a subsequent pharmacophoric search using Unity (SYBYL 7.0, Tripos Inc., St. Louis, Missouri, 63144, USA).

The receptors used for the Unity search were the crystal structures 1 UK4 (SARS-CoV M^pro in complex with the irreversible pentapeptidyl chloromethyl ketone NSTLQ-CH₂-S-Cys145 (SEQ ID No. 4: NSTLQ) and 1 UJ1 (free enzyme at pH 6.0). Finally, as result of the diversity analysis and pharmacophoric unity search a subset of 234 compounds was selected from the 3,000 nucleophilic fragments.

It is understood, that this is just an example and that every other library or collection of compounds can be used.

Example 15: Screening for bioactivity of nucleophilic fragments

The nucleophilic fragments were tested for their bioacitivity against SARS-CoV M^pro in our functional enzyme assay based on the fluorogenic substrate AcTSAVLQiAMCA. Cleavage reactions were incubated at 298 K and contained 1 μM SARS-CoV M^pro, 1 mM of Ac- TSVALQ-AMCA, and 400 μM of a nucleophilic fragment in a total volume of 20 μl 100 mM MES pH 7.0 in a 384-well microtiter plate. The initial rate was observed and compared with the initial rate without any nucleophilic fragment.

The use of the method for the target SA RS-CoV M^pro is just one example. The invention can be carried out with all the targets described in the application and is not limited to SARS-CoV M^pro.

Example 16: Method of the invention used for P1 The method was performed using 1 μM of SARS-CoV M^pro, 1 mM of Ac-TSVALQ J.AMCA, 400 μM of a nucleophilic fragment, and 50 μM of the peptide aldehyde inhibitor Ac-DSFDQ-H in a total volume of 20 μl 100 mM MES pH 7.0 in a 384-well microtiter plate. The initial rate was observed and compared with the initial rate of the controls.

Example 17: Method of the invention used for P1 ^'

Target Induced Formation Screening for the PV moiety was performed using 1 μM of SARS- CoV M^pro, 200 μM of Ac-TSVALQ jAMCA, 200 μM of a nucleophilic fragment and 5 μM of the non-peptidic inhibitor 17 in a total volume of 20 μl MES (100 mM, pH 7.0) in a 384-well micro- titer plate. The initial rate was observed and compared with the initial rate of the controls.

Example 18: Bioassay with SARS-CoV M^pr0

The inhibitory activities found were validated in the established HPLC-based enzyme assay by Tan et al (2005, J. MoI Biol). The sequence of peptide substrate used was SWTSAVLQlSGFRKWA-NH₂. (SEQ ID NO. 5: SWTSAVLQSGFRKWA) Cleavage reactions were incubated at 298 K and contained 1 μM SARS-CoV M^pro, 100 mM MES pH 7.0 and 0.5 mM peptide substrate in a total volume of 20 μl. To stop the enzyme reaction, 80 μl of 2% TFA was added and being stored at 193 K. The samples were centrifuged for 10 min at 15,000 g before analysis by reverse-phase HPLC on a C18 column (3.9 x 150 mm). Cleav- age products were resolved by using a 15 min, 5-60% linear gradient of acetonitrile in 0.1% trifluoroacetic acid. The absorbance was determined at 280 nm, and peak areas were calculated by integration.

Example 19: Synthesis and kinetic analysis of the fluorogenic substrate Ac-TSAVLQ- AMCA.

For detection of inhibition, a fluorescence-based functional bioassay was developed. The fluorogenic substrate Ac-TSAVLQ-AM CA 1 was synthesized by using the bifunctional fluoro- phore 7-amino-4-methyl-3-coumarinylacetic acid (AMCA) as described. A yield of 90% was observed. The activity of SARS-CoV M^pro was determined by measuring the release of AMCA (see figure 2). The functional enzyme assay was performed in a total volume of 20 μl in 384-well microtiter plates and is thus suitable for high-throughput screening (HTS). The K_M and V_max values were determined by measuring and plotting the initial rate, V, over a range of substrate concentrations S (0.25 - 2.5 mM), and fitting the data directly to the Lineweaver-Burk diagram (not shown).

The double-reciprocal plot gave a V_max of 9.06 μM/min and a K_M of 210 μM for Ac-TSAVLQ- AMCA 1. From there, we calculated a k∞x of 9.06 min^"1 (an enzyme concentration of 1 μM was used) and a /C^/K_M value of 43.3 mM^"1 min^"1. Our recombinant SARS-CoV M^pro contained a C-terminal His-tag. Tan el al. (2005, J. MoI Biol) reported for this enzyme construct a /WK_M of 61.9 mM^'1 min^"1, determined in an HPLC assay with an extended substrate peptide. This agrees with most of the literature on proteases. Substrates with AMCA in PV should be relatively poor substrates, because this bulky group should be difficult to accommodate in the rather restricted SVsite.

234 nucleophiles were selected from a larger fragment library. For control, all selected frag- ments were tested for bioactivity against SARS-CoV M^pro in the functional enzyme assay described above at concentrations of 400 μM. None of the selected fragments was found active as SARS-CoV M^pro inhibitor at this concentration. As the site-directing, electrophilic probe, the peptide aldehyde Ac-DSFDQ-H 2 was selected due to its moderate affinity and its good water solubility. All fragments were then incubated with the enzyme SARS-CoV M^pro, the fluorogenic substrate Ac-TSAVLQ-AMCA 1, and the selected peptide aldehyde inhibitor Ac-DSFDQ-H 2. Cleavage of the fluorophore 7-amino-4-methyl-3-coumarinylacetic amide (AMCA) was recorded over time at 460 nm. The initial rate of product formation in the presence of peptide aldehyde 2 and the nucleophilic fragment was determined by linear fitting of the data and compared to the uninhibited enzyme reaction and to the inhibited reaction with- out the addition of nucleophilic fragments (see figure 3).

Figure 3: For monitoring the enzymatic reaction, cleavage of the substrate 1 was recorded over time by excitation at 380 nm and emission at 460 nm for released AMCA. The inhibitory effect of fragment 3 was detected as a significant decrease in the rate of the enzyme reaction compared to the negative control (no inhibitor) and to the positive control (peptide aldehyde inhibitor Ac-DSFDQ-H 2 without the addition of a nucleophilic fragment).

For 227 nucleophilic fragments the observed initial rates of product formation were higher than or equal to those observed for the peptide aldehyde alone. This finding indicates that the 227 inactive fragments do not form an inhibitory ligation product by reaction with the pep- tide aldehyde 2. In some cases, inactive nucleophiles completely abolished the inhibition of peptide aldehyde 2 indicating that the ligation reaction completely removes the peptide aldehyde from the active site of the protease.

In seven cases, a significantly reduced initial rate of the enzyme reaction and, consequently, a stronger inhibition than with the inhibitor 2 alone was observed.

It remained to be demonstrated that the inhibition of SARS-CoV M^pro was effected by the ligation product of 2 and the respective nucleophile and that the active fragments actually bind to the active site of the protease. The active hit-molecule 3 was selected for exemplary verification of the inhibition by means of synthetic fragment derivatives. The imine is the hy- pothesized ligation product of peptide aldehyde 2 with compound 3. In order to collect evidence for the site-directed binding of fragment 3, at first the reduced ligation product Ac- DFSDQ(ψCH₂-NH)-(3-amino-Λ/-(3-amino-phenyl)-benzamide (10) was synthesized, and tested in the HPLC-assay described earlier by Tan et al. (2005, J. MoI Biol) 10 displayed a K, of 50.3 μM. Comparison of this activity with the completely inactive reduced amide 11 and likewise with the inactive peptides Ac-DSFDQ-OH (12), DSFDQ-OH (13), and Ac-DSFDQ- NH₂ (14) indicated clearly the directing effect of the tetrapeptide and suggested the binding of fragment 3 to the S1 ^'-site. The reduced inhibition relative to the peptide aldehyde 2 can be explained by deletion of the electrophilic site that should interact favorably with the active site cysteine of SARS-CoV M^pro

Additional evidence for the binding of fragment 3 in the S1 ^'-pocket should be available by the design and synthesis of derivatives of 3 bearing an electrophilic warhead and thus should be capable of interacting with the active site of the protease. The electrophilic derivatives of 3, i.e. compounds 15, 16, 17, and 18, which only differ in the position of the aldehyde function relative to the phenyl ring, showed K₁ values of 19.9 μM (compound 15), 33.5 μM (compound 16), 14.7 μM (compound 17), and 50.3 μM (compound 18), respectively (HPLC-based as- say). The lower K_\ values of 15 and 17 versus 16 and 18 can be rationalized by two arguments. First, in compounds 15 and 17, the aldehyde electrophile is positioned isosterically to the electrophilic carbon in the peptide aldehyde 2 and the imine derived thereof, whereas in compound 16 the position of the electrophile is shifted relative to the putative imine ligation product. Secondly, a benzaldehyde (16) and a phenylglyoxal (18) are less electrophilic than their aliphatic equivalents due to mesomeric stabilization by the aromatic system. Generally, from these examples it can be seen that the electrophilicity is essential for the activity of these inhibitors. Whereas the nucleophilic fragment 3 alone is nearly inactive though it has an affinity to the SV pocket of the SARS-CoV enzyme, the additional interaction of an electrophile in compound 15 with the nucleophilic thiol of the cysteine residue in the active site, increases the affinity of the nucleophilic fragment significantly, presumably due to formation of a tetrahedral transition state isoster. In complete agreement with this transition-state concept, the reduced peptide amide Ac-DSFDQ-(ΨCH₂-NH₂) (11) and the analogous peptide acids Ac-DSFDQ-OH (12), DSFDQ-OH (13), and Ac-DSFDQ-NH₂ (14) were inactive at a concentration of 500 μM. In addition, for another negative control benzaldehyde 23 alone was inactive as well (>500 μM). In summary, based on the observed K\ values of the electrophilic fragments, the peptides and the reductive amination products; it is conceivable that the found fragments have an affinity for the ST pocket of the M^pro.

Targeting the S 1 -site To obtain an entirely non-peptidic inhibitor of SARS-CoV M^pro targeting both the S^'1 and S1 pockets, the 2-keto aldehyde 17 was employed as a directing probe in a second Dynamic Ligation Screening. This time, we screened 110 nucleophilic fragments that we had selected by diversity analysis. Compound 17 was also incubated with one nucleophilic fragment per well and the protease. Then the fluorogenic substrate Ac-TSAVLQ-AMCA 1 (SEQ ID No. 3: TSAVLQ) was added and we observed the formation of free AMCA by fluorescence detection. In the second screen, three active fragments were identified that are able to enhance inhibitory activity in presence of the directing probe 17.

The choice of 2-keto aldehydes as directing probes in the second round should allow the synthesis of a covalent coupling product by reductive amination with the active nucleophile under retention of the keto functionality as an electrophile. For example, reductive amination of 2-keto aldehyde 18 and the amine 19 yielded the 2-amino ketone 22 employing trichlorosi- lane as reductive agent. 22 was tested against M^pr0 in the HPLC-assay and was found to be active with a K_\ value of 2.9 μM. Thus, starting from the peptidic inhibitor Ac-DSFDQ-H 2 with a K_\ of 10.9 μM, we could identify 7 fragments with low affinity for the SV pocket. One hit was selected (3) and, after introduction of a bis-electrophile, served as a site-directing probe for a second Dynamic Ligation Screening. Combination of two low-affinity fragments into one molecule finally furnished the non-peptidic inhibitor 22 with a K_\ value of 2.9 μM.

Claims

Claims

1. Method for a rapid and sensitive identification of binding and/or activity of low-affinity compounds for an active site of a target, wherein a formation of a binding-species comprising one of the said compounds and an electrophilic binder preferred via a reversible ligation reaction is detected in a parallel detection assay.

2. Method, preferred of claim 1 , for an identification and/or production of a binder for an active site of a target, wherein the method comprises the following steps:

a) providing the target,

b) addition of an identified and/or synthesized electrophilic binder, preferred a compound selected from table 1 , especially an aldehyde, which binds to the active site of the target,

c) addition of a first compound, selected from the group of low molecular weight compounds, in particular nucleophiles, preferred amines, thiols, alco- hols, hydrazines and/or hydroxylamines especially preferred selected from the group comprising the compounds of table 2,

d) detection of the binding, preferred a reversible binding, of a first binding- species to the target in a parallel detection assay, wherein the first binding-species is a product of a ligation, preferred a reversible ligation, of the first compound and the electrophilic binder,

and wherein the binder is the first compound and/or the first binding-species.

3. Method according to claim 1 or 2, wherein the method comprises a modification of the identified first compound, preferred an introduction of an electrophilic warhead whereby a modified second compound is obtained, preferred selected from the group comprising the compounds of table 1.

4. Method according to at least one of the preceding claims, further comprising the following steps:

e) providing the target,

f) addition of the modified second compound and/or a derivative thereof, g) addition of the first compound and/or a third compound selected from the group of low molecular weight compounds, in particular nucleophiles, preferred amines, thiols, alcohols, hydrazines and/or hydroxylamines especially preferred selected from the group comprising the compounds of table 2,

h) detection of a binding, preferred a reversible binding, of a second binding- species to the target in a parallel detection assay, wherein the second binding-species is a product of a ligation, preferred a re- versible ligation, of the third compounds and/or the first compound and the modified second compound and/or a derivative thereof,

and wherein the third compound and/or the first compound and/or the second binding- species are identified and/or produced, preferred wherein the second binding-species is non-peptidic, preferred selected from the group comprising the compounds of table 3.

5. Method according to at least one of the preceding claims, wherein the method further comprises a production of the second binding-species and/or derivatives thereof, wherein the modified second compound and/or a derivative thereof is ligated to the identified first compound and/or the identified third compound and/or derivatives thereof.

6. Method according one of the preceding claims, wherein the electrophilic binder and/or the modified second compound and/or a derivative thereof are used as an active site directing probe.

7. Method according one of the preceding claims, wherein the first and/or the third compound are low-affinity binder, binding to the target with K_D > 100μM.

8. Method according to at least one of the preceding claims, wherein the detection assay is a enzyme assay and/or a protein assay, preferred wherein the binding of the first binding-species and/or the second binding- species and/or derivatives thereof to the target is determined via an amount of product formation in the enzyme assay and/or an fluores- cence signal, fluorescence polarization signal, luminescence signal,

UV/VIS absorption and/or radioactive signal in a protein assay preferred an ELISA or fluorescence polarization assay.

9. Method according to at least one of the preceding claims, wherein a substrate of the target is added, preferred a labeled substrate, preferred with a reporter molecule and wherein the substrate and the first binding-species or the second binding- species cannot bind to the target at the same time, preferred wherein they compete for the same active site of the target.

10. Method according to at least one of the preceding claims, wherein the electrophilic binder, preferred selected from table 1 , especially an aldehyde, is a substrate of the target, preferred a labeled substrate.

11. Method according to at least one of the preceding claims, wherein the target is a macromolecule, selected from the group comprising a protein, an enzyme, a biological receptor, an antibody, a ribonucleic acid molecule and/or a deoxyribonucleic acid molecule.

12. Method according to at least one of the preceding claims, wherein the enzyme is selected from the group comprising SARS coronavirus main protease, phosphotyrosine phosphates, preferred ptp1 B, MptpA, SHP-2, ptpN7, ptpRR, phosphotyrosine binding proteins, preferred STAT-5, human immunodeficiency virus protease-1 and/or β-amyloid converting enzyme-1.

13. Method according to at least one of the preceding claims, wherein the electrophilic binder is selected from the group comprising the compounds of table 1 , aldehydes, ketones, carboxylic acid ester, carboxylic acid thioester, imines, iminium salts, halogenides, epoxides and/or elektrophilic olefins, preferred Ac-DSFDQ-H, carboxyfluoresceine DEVD-CO-CHO, Ac-DEVD-CO- CHO, 4-Formyl-phenyl-phosphate, 4-formylphenyl dihydrogen phosphate, the compound of formula 2, the compound of formula 3 and/or the compound of formula 4.

14. Method according to at least one of the preceding claims, wherein the detection assay comprises an enzymatic detection via a release of a re- porter molecule, preferred a fluorescent reporter molecule and/or a protein binding assay performed by fluorescence, fluorescence polarization, luminescence, UVΛ/is absorption or radioactive signal like fluorescence polarization assay or ELISA.

15. Method according to at least one of the preceding claims, wherein the reporter molecule is 7-amino-4-methyl-3-coumarinylacetic amide and/or a carboxyfluoresceine labelled binding molecule preferred carboxyfluoresceine- DEVD-CO-CHO or an absorbing or fluorescent heavy metal phosphate complex.

16. Method according to at least one of the preceding claims, wherein the detection assay is performed as a high-throughput assay, preferred in mi- crotiter plates.

17. Method according to at least one of the preceding claims, wherein the method is performed as a high-throughput method, preferred in microtiter plates.

18. Method according to at least one of the preceding claims, wherein the second binding-species comprises a molecule of formula 1 :

(1)

19. Method according to at least one of the preceding claims, further comprising formulating the first compound, the second compound, the third compound, the first binding- species and/or the second binding-species and/or derivatives thereof in a pharmaceutically acceptable form.

20. A Method for the production of a pharmaceutical composition comprising the method of at least one of the preceding claims and furthermore mixing the first compound, the second compound, the third compound, the first binding-species and/or the second binding-species and/or derivatives thereof with a pharmaceutically acceptable carrier.

21. Method according to the preceding claim, wherein the carrier is selected from the group comprising fillers, disintegrants, binders, humectants, extenders, dissolution retarders, absorption enhancers, wetting agents, adsorbents and/or lubricants.

22. The first Compound, the second compound, the third compound, the first binding- species, the second binding-species and/or derivatives thereof produced by the method according to any of the preceding claims.

23. The second binding-species, preferred according to formula 1 , the second compound, the first binding-species and/or derivatives thereof produced by the method according to any of the preceding claims.

24. A pharmaceutical agent comprising the first compound, the second compound, the third compound, the first binding-species, the second binding-species and/or derivatives thereof and a pharmaceutically acceptable carrier.

25. The pharmaceutical agent according to the preceding claim, characterized in that said agent is a capsule, a tablet, a coated tablet, a suppository, an ointment, a cream, an injection solution, an infusion solution, a vaginal and/or rectal suppository, a pad and/or a foam.