Fragment based drug discovery (FBDD) has emerged as a powerful approach for the design and identification of new drug leads.17 It employs biophysical and structural assays for the discovery of weakly but specifically binding low molecular weight (MW < 300 Da) molecules (fragments) that can be further optimized to bigger drug-like molecules by adding functional groups or linking or merging several fragments.18 Screening a library of small-molecules poses several advantages over conventional high-throughput screening approach. FBDD is more efficient as it allows to screen a smaller library consisting of structurally and chemically diverse fragments that cover a broad chemical space.19 Due to the small size, fragments pose a lower degree of complexity and are therefore less likely to form unfavorable interactions.
In addition, they can bind to region that are difficult to access by bigger molecules.19 Fragment-based drug discovery has been successfully employed in targeting PPIs. The majority of small-molecules on the market are PPI inhibitors.20 Chapter 2 and 4 employed the opposite strategy by exploring the potential of small-molecule induced stabilization of cancer associated 14-3-3σ – p53 PPI. A natural compound Fusicoccin-A (FC-A) proved to be a good tool compound for screening small-molecule PPI stabilizers. It induced a 2-fold stabilization of 14-3-3σ – p53 PPI as described in Chapter 2. To further prove the importance of rational peptide design and selection in drug discovery, the FC-A induced stabilization effect on different lengths of p53 peptides was measured using surface plasmon resonance (SPR) assay. Comparison of the binding affinities of 15mer and 20mer peptides showed that the addition of FC-A led to approximately 2-fold stabilization of the 14-3-3σ – peptide interaction in accordance with previously reported data (Figure 6.1). Interestingly, 12mer peptide which showed no binding in the absence of FC-A, bound to 14-3-3 with a Kd of around 55.7 µM in the presence of the compound. In addition, 14mer that has a relatively weak binding affinity to 14-3-3σ, showed a 3-fold stabilization effect upon the addition of FC-A.
Figure 6.1. Fusicoccin-A induced stabilization effect on the binding affinity of different lengths of p53 peptides to 14-3-3.
Although Fusicoccin-A serves a good positive control for the identification of small-molecule 14-3-3σ – p53 modulators, it also sheds light on the challenges that need to be overcome when designing and screening PPI stabilizers. One of the most important key features of a successful fragment hit is its specificity. FC-A has been shown to interact with and stabilize several 14-3-3 protein-protein interactions.21 Further optimization of FC-A could improve its specificity. However, synthesize of Fucicoccin-A analogues has proven the be synthetically challenging. Therefore, additional approaches for the discovery of 14-3-3 PPI stabilizers are needed.
Detailed structural information of a fragment binding site at molecular level significantly facilitates rational design of small-molecule stabilizers or inhibitors.19 Chapter 4 described a crystallography-based fragment design for the identification of 14-3-3σ – p53 PPI stabilizers.
In order to achieve a specificity, the unusual binding mode of p53 peptide has to be taken into consideration. Due to the unique amino acid sequence the C-terminal domain of p53 peptide occupies only 2/3 of the ligand binding pocket. Small-molecule stabilizers can bind to the remaining space and form interactions with Glu-388 of p53 that is directed towards the binding channel. These structural characteristics enable to design fragments that can distinguish between the U-shaped binding mode of the tumor suppressor protein p53 from proteins that bind to 14-3-3 in an extended conformation such as a transcriptional co-activator TAZ (Figure 6.2 A).22 Hence, the fragments targeting 14-3-3σ – p53 PPI interface
were designed to bind in the shallow pocket in a desirable conformation and to make contacts with the carboxyl group of Glu-388.
Figure 6.2. (A) Superimposition of the crystal structures of the p53pT387(pink sticks) and TAZpS89 (blue sticks) (PDB 5N75) peptides bound to 14-3-3σ (gray cartoon). (B) Structure of AZ-022. The amine-terminated functional group is surrounded by a red circle.
Although, co-crystallization trials of a potential small-molecule stabilizer AZ-022 were not successful, docking studies predicted the flexible amine-terminated side-chain of the fragment to reach towards the p53 peptide and form a salt bridge with Glu-388. Since the TAZ peptide occupies the entire binding channel, AZ-022 is not expected to bind to 14-3-3σ – TAZ interface. Although NMR, FP and SPR experiments all confirmed the AZ-022 induced stabilization effect, further fragment design is necessary for the development of a potent 14-3-3σ – p53 PPI stabilizer. An extension of the amine-terminated functional group of AZ-022 to reach the p53 peptide would be a good starting point (Figure 6.2 B). A docking pose of ten such fragments have been proposed and the synthesize of these fragments is currently ongoing.
Structural studies have revealed that most of the proteins have several small-molecule binding sites.23 This was further proved by fragment-screening approach that led to the identification of two secondary binding sites on 14-3-3 that are located on top of 14-3-3 monomers.22 In Chapter 5, three more of these sites were described. Of them two are located at the 14-3-3 dimerization interface. 14-3-3 exerts many of its important functions as a homodimer.24 Therefore, small-molecule induced dissociation or association of dimers
can be used to modulate 3 activity in disease related processes. Considering that 14-3-3 isoforms share a highly conserved amphiphatic ligand binding groove, targeting the dimerization interface that differs between isoforms, may be a promising approach for isoform specific targeting of 14-3-3 proteins.24 Increased expression of 14-3-3 has been reported in several human cancers and cause resistance to anti-cancer treatment.25,26 Inhibition of dimerization disrupts the 14-3-3 regulated resistance to drug-induced apoptosis and is a promising approach for sensitizing cancer cells to anti-cancer treatment.24,27 This thesis described the discovery of dimerization stabilizers and inhibitors that could serve a good starting point for further optimization of isoform specific PPI modulators. However, additional experiments are needed to evaluate the isoforms specific activity of identified fragments. In addition, further structural studies would help to shed light on the binding mode of the fragments and provide useful information for subsequent rational fragment design.
Conclusion
PPI modulation is a powerful approach in drug discovery. In addition to conventional approaches, PPI stabilization has a great potential in targeting disease related processes.
However, detailed molecular characterization of PPI is essential for the design of biophysical assays for identification of novel and selective PPI modulators. Combining fragment-based drug discovery with knowledge of the molecular mechanism of the PPI enables a rational design of selective PPI modulators. This thesis aimed to describe the potential of such approach and provides a starting point for the development of PPI modulators.
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