Irreversible mono-ligand inhibitors

Irreversible inhibitors form a covalent bond (or several covalent bonds) upon binding to the kinase, and hence cannot be displaced by other inhibitors or substrates. According to the chemical properties, irreversible inhibitors can be divided into two groups: the compounds carrying reactive chemical groups (usually electrophiles) that instantly form a covalent bond with the PK upon binding, and compounds possessing so-called photo-affinity groups (usually aromatic azides or benzophenones) that serve as reversible inhibitors in the dark but become reactive upon UV-irradiation. The first irreversible inhibitors designed for kinases were ATP and cAMP analogues labeled with radioactive isotopes and incorporating either a photo-affinity azide-group or a chemically reactive p-fluorosulfonylbenzoyl moiety [Chuan et al., 1989; Kerlavage and Taylor, 1980; Zoller et al., 1981] (Figure 7A). These compounds were used for determination of residues in the kinase sites responsible for binding ATP or cAMP, respectively; after covalent modification, the kinases were subjected to proteolytic cleavage, and the radioactively labeled residues were isolated.

Later on, a similar approach named activity-based protein profiling (ABPP) was introduced, based on irreversible inhibitors termed activity-based probes (ABPs). An ABP consists of three fragments: a moiety endowed with affinity towards protein kinase(s) of interest, a chemical group responsible for gene-ration of a covalent bond, and a reporter-tag for the detection of covalently modified PK (i.e., a radioactive isotope, fluorescent dye, or biotin moiety) [Rix and Superti-Furga, 2009; Speers and Cravatt, 2004]. ABPs selective towards certain PKs have been used to “catch” the target protein(s) from the biological samples (i.e., cell or tissue lysates), or to generate an affinity matrix where natural ligands interacting with enzyme (but not binding to the same site as ABP) may bind [Gayani et al., 2008; Kalesh et al., 2010]. Additional application possibilities are available for the generic ABPs, i.e., those lacking selectivity within a certain group of PKs or most of the kinome). The latter may be used for identification of kinomic pattern of biological samples (so-called chemical proteomics), or for assessment of affinity and selectivity of non-covalent inhibitors added to the PK-containing sample prior to introduction of ABP [Ratcliffe et al., 2007; Speers and Cravatt, 2004]. Apart from kinases, ABPs have been generated for over 20 enzyme classes, including major families of proteases, phosphatases, glycosidases and glutathione S-transferases, and a range of oxidoreductases [Saghatelian and Cravatt, 2005].

Another variant of irreversible inhibitors is represented by cross-linking compounds, which incorporate an affinity-fragment responsible for binding of compound to protein and two reactive groups responsible for covalent linking.

One of the reactive groups generates a covalent bond with the PK (usually at the ATP-site), and the other reactive group forms a covalent bond with the protein or peptide binding to the substrate-binding site of the same PK [Liu K. et al., 2008; Parang et al., 2002b]. A cross-linking compound should therefore protrude into the substrate-site to modify covalently one of the protein/peptide

residues but not deep enough to interfere with its binding. Dialdehyde moieties have been frequently used as electrophilic moieties able to react irreversibly with nucleophilic groups of amino acid side-chains incorporated in the structure of kinase and substrate (Figure 7B). While in PKs, the reactive nucleophilic group is incorporated in the side-chain of the highly conserved Lys residue (corresponding to Lys72 in PKAc), the range of substrates suitable for trapping into cross-linked PK-substrate complexes is limited with Cys-containing pseudosubstrate-peptides [Kalesh et al., 2010; Liu K. et al., 2008; Maly et al., 2004; Statsuk et al., 2008]. Thus, dialdehyde-containing cross-linking com-pounds cannot be used for identification of novel kinase substrates in biological samples, but rather for selective detection of kinase itself, whereas selectivity may be achieved by use of a selective pseudosubstrate and/or a selective cross-linking compound. As an alternative, cross-cross-linking compounds have been developed incorporating two azide groups that are both converted to reactive nitrene or diazaquinodimethane intermediates upon UV-irradiation (Figure 7C) [Parang et al., 2002b; Polshakov, et al. 2005]. These intermediates are able to react with O-H and C-H bonds of proteins, and hence do not necessarily require presence of strong nucleophiles in the neighborhood of their binding site in order to form covalent bonds [Matheson et al., 1977].

A B

C

Figure 7. Examples of irreversible inhibitors. (A) FSBA [Ratcliffe et al., 2007]. (B) Dialdehyde 7 [Kalesh et al., 2010]. (C) Bifunctional azide-based crosslinker 5 [Parang et al., 2002b].

The problematic issue of all aforementioned classes of irreversible inhibitors is loss of affinity and/or selectivity of the PK-binding fragment upon conjugation with reactive groups responsible for forming the covalent bonds. However, several irreversible inhibitors have been developed possessing sufficient affinity and selectivity for application as drugs (i.e., for the treatment of non-small cell

N

lung cancer and other types of solid tumors). Most of the irreversible inhibitors undergoing clinical trials are targeted to ATP-sites of EGFR or HER2 and represent derivatives of quinazoline, anilinoquinoline, pyridopyrimidine, or pyrimidine incorporating unsaturated Michael acceptor groups that form covalent bond with Cys residues of tyrosine kinases (i.e., Cys773 in EGFR, Cys805 in HER2) [Fry, 1999; Heymach et al., 2006; Smaill et al., 1999; Zhang J. et al., 2009; Zhou W. et al., 2009]. Recently, similar compounds targeting Bruton's tyrosine kinase and VEGFR-2 have been reported (Figure 8A). In the latter case, derivatives of quinazoline were used containing two reactive groups positioned in such way that one (Michael acceptor) generates a covalent bond with EGFR or HER2 and the other (benzoquinone moiety) with VEGFR-2 [García-Echeverría et al., 2000; Wissner et al., 2007; Zhang J. et al., 2009].

Another selectivity-gaining binding mode was utilized by the natural compound lactoquinomycin, which formed covalent bonds with the residues Cys296 and Cys310 of the activation loop of PKB [García-Echeverría and Sellers, 2008;

Toral-Barza et al., 2007]. Lactoquinomycin showed significant selectivity towards PKB in a panel of 45 PKs and inhibited PKB-catalyzed phospho-rylation in cells; however, the nonspecific redox-related cytotoxicity of this compound strongly limited its pharmacological applications. Last but not least, pyrrolopyrimidine derivatives containing either a chloromethylketone (CMK) or a fluoromethylketone (FMK) electrophile could also be successfully applied for irreversible inhibition of RSK isoforms 1, 2 and 4, and showed remarkable target selectivity not only in biochemical in vitro systems, but also in mam-malian cells [Figure 8B; Cohen M. S. et al., 2005]. The latter observation might be attributed to the incorporation of a second selectivity determinant into the structure of compounds, represented by a para-tolyl substituent targeted to the hydrophobic pocket at the back of the ATP-site that is lined by a compact gate-keeper residue (Thr) in case of RSK isoforms 1, 2 and 4.

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Figure 8. Examples of selective irreversible inhibitors. (A) Compound 17 [Wissner et al., 2007]. (B) CMK and FMK; selectivity determinant marked with red oval [Cohen M.

S. et al., 2005].

To sum up, it should be mentioned that all irreversible inhibitors exhibit both time- and concentration-dependent manner of PK inactivation [Fry, 1999].

Moreover, in most cases the results of measurements of affinity and inhibition potency of irreversible inhibitors are dependent on the rate of formation of covalent bond with a kinase, and in case of chemically reactive compounds may even reflect affinity of both reversibly and irreversibly binding forms. The most problematic issue for the development of irreversible inhibitors remains exact identification of the positions in the inhibitor structure where reactive groups should be positioned to generate covalent binding to the protein residues. For irreversible inhibitors applied in crude biological systems, a problem of exces-sive activity may also evolve, i.e., irreversible binding to the compounds containing nucleophilic centers and present in cell in high concentration (i.e., glutathione) [Wissner et al., 2007; Zhang X. et al., 2008]. In case of pharma-cological applications, the issue of selectivity might become especially important, as a covalent attachment to unanticipated targets should by all means be avoided to reduce the toxicity of irreversible inhibitors. The advantages of irreversible compounds, however, sometimes outweigh the potential risks, as covalent inhibitors ensure prolonged inhibition of enzyme and do not need to achieve high plasma concentrations in order to gain inhibitory potency [Fry, 1999; Morphy, 2010]. Moreover, ATP-site directed irreversible inhibitors do not suffer as much from the high intracellular concentrations of ATP as their reversible counterparts: once the covalent bond is formed between an inhibitor and a PK, the former cannot be displaced from the compex. Hence, covalently binding compounds are also of especial value for inhibition of proteins that acquire high affinity towards ATP as a result of mutation [Fry, 1999; Krishna-murty and Maly, 2010].