1.3.1. Small molecule Integrin inhibitors
The fact, that Integrins are involved in many pathological situations like cancer, thrombosis or immune disorders makes Integrin-signaling an interesting target for inhibitors (Hynes.2002; Winograd-Katz et al., 2014). The first Integrin-targeting drugs were developed to treat thrombosis and function by inhibiting the Integrin αIIbβ3-fibrinogen binding that is responsible for platelet-platelet interactions during thrombus formation (Coller and Shattil, 2008). This inhibition of ligand-binding is achieved either by β3 specific antibodies, small RGD-like peptides or small molecules that act as competitive inhibitors. Tirofiban is an example for such a small molecule inhibitor (Fig. 1.8A). This inhibitor was developed on the basis of the viper venom peptide, echistatin and is approved for the treatment of acute coronary syndromes (Bledzka et al., 2013). Most of the inhibitors that are currently used to interfere with Integrin signaling target the ligand-binding, extracellular part or modulate Integrin expression (Cox et al., 2010). Novel strategies involve the usage of peptides or small molecules that specifically inhibit the interaction of Integrin cytoplasmic tails with their binding partners. One such molecule is the small molecule compound 6-B345TTQ that is able to disrupt binding of Paxillin to the cytoplasmic tail of Integrin α4 (Fig. 1.8A), an interaction that was shown to be important for immune cell migration (Liu et al., 1999; Rose, 2006). A proof of principle study demonstrates the inhibitory potential of this molecule to inhibit T-cell migration and its potency to be used as a strategy to reduce inflammation (Kummer et al., 2010). In contrast to the few available small molecules that inhibit Integrin signaling, an emerging number of protein-protein interactions within the Integrin adhesome are implicated in pathological processes and therefore strongly suggested as potential therapeutic targets. One such important protein-protein interaction within the Integrin signaling pathway is the binding of FAK to Paxillin that is crucial for FAK recruitment to FAs and FAK-mediated signaling (Deramaudt et al., 2014). Alterations of this interaction reduce cell migration and invasion and thus make the FAK-Paxillin interaction an interesting target for the development of small molecule inhibitors (Deramaudt et al., 2014) Inhibition of Integrin signaling at the
27 level of downstream cytoplasmic proteins may enable a new specificity for particular Integrin functions while sparing others (Cantor et al., 2008).
1.3.2. Phenotypic high content screen and target identification
Clearly, a variety of interactions within focal adhesions may serve as a potential therapeutic target. One strategy to identify new compounds that disrupt protein-protein interactions and influence the subcellular localization of a protein-protein of interest is the use of high content screens (HCS) that are based on microscopy techniques (Fig. 1.8B). This procedure involves automated cell-seeding and compound transfer as well as image acquisition and analysis (Kau et al., 2004). After the successful identification of small molecules that are responsible for a desired phenotype, the molecular target needs to be determined. For this purpose, a variety of methods are available ranging from next-generation sequencing, radiolabeling of molecules to the affinity-based purification of target proteins (Farha and Brown, 2016). Among them, the affinity-based approach can be applied to a variety of experimental questions and is the most frequently used method for target identification. More specifically, the compound is either directly conjugated to a resin (e.g. agarose) or linked to an affinity moiety such as biotin and incubated with cell extracts containing the putative target(s) (Fig. 1.8C, I.-II.). The purified proteins can then be eluted and identified for example via mass spectrometry. In addition to the classical approaches, there are several improvements and modifications that might facilitate the experimental procedure and/or enable a more specific purification strategy (Fig.
1.8C, III.-VI.). For example, magnetic particles can be easily dispersed and recovered and have the advantage of a large surface area as well as resistance to organic solvents (Kawatani and Osada, 2014). The use of a rather bulky tag like biotin often has influence on the activity of the compound and prevents the purification of targets from living cells (Kawatani and Osada, 2014). To circumvent this problem, bioorthogonal chemistry can be applied. This method uses the copper-catalyzed Huisgen-azide-alkyne cyclo-addition (click chemistry) to conjugate a functional tag at the desired step of the purification procedure (Fig. 1.8C). Such bioorthogonal molecules can be further improved by the addition of a photoreactive group, like benzophenone (Lapinsky, 2012). These modifications lead to trifunctional probes that enable a covalent crosslinking of the small molecule and its
28 target, which makes the complex stable throughout the purification process (Kawatani and Osada, 2014).
Although the affinity-based approaches represent a straight forward method it contains many steps that influence success or failure in target identification. Critical steps are for example the design of the probe and the determination of possible modifications that do not alter the compounds activity. Furthermore, linker type and length as well as the used buffers may influence non-specific binding and/or the accessibility of the target protein.
Considering the described (and also all unmentioned) obstacles that can arise during affinity-based target identification, it becomes clear that this method might demand a relatively high effort and could tremendously increase the time period between the initial phenotypic screen and the determination of the molecular target.
This could, at least partially, explain that several studies report the identification of compounds through HCS without providing a detailed knowledge of the molecular targets. For instance, Peppard et al (2015) described a phenotypic screen for compounds specifically stimulating the differentiation of oligodendrocyte precursor cells that can serve as a basis of the treatment of multiple sclerosis. They were able to identify 22 interesting compounds which need to be analyzed further in regard to their detailed biological activity (Peppard et al., 2015). Another fluorescence microscopy based screen, identified small molecule inhibitors of the Rho pathway, termed Rhodblocks (Castoreno et al., 2010). The group of Rhodblocks encompasses eight compounds that inhibit cytokinesis but might have different molecular targets along the pathway. Despite the lack of knowledge on the direct molecular target, the complexity and the set-up of such HCS as well as the functional relevance of the involved signaling pathway are of special interest. Due to this, such studies contribute strongly to the improvement of microscope-based screening procedures, which will help to identify new small molecule inhibitors.
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Figure 1.6: (A) Small molecule compounds that inhibit Integrin signaling. Tirofiban functions as competitive inhibitor for the fibrinogen binding of Integrin αIIbβ3. 6-B345TTQ represents a novel strategy for inhibition of Integrin signaling by disrupting the Integrin alpha4-Paxillin interaction (taken from Cox et al., 2010). (B) Workflow of a microscope-based high-content screen (taken from Kau et al., 2004). (C) Strategies for the affinity-based target identification. I. The small molecule is immobilized and used for purification of target proteins that can finally be identified using mass spectrometry. Immobilization can be directly on an agarose resin (II.) on a streptavidin resin using a biotin tag (III.) or on magnetic nanoparticles (VI.). For in situ labeling with minimal structural perturbations compounds can be modified with a bioorthogonal tag alone or within a trifunctional probe to enable covalent crosslinking between the small molecule and the target protein. (taken from Kawatani and Osada, 2014).
A B
C
I. II. V.III.
VI. VI.
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