Protein-protein interactions (PPIs) are central to a large number of vital biological processes and thus represent attractive targets for the development of novel therapies for a variety of diseases.(1
) Although scientists recognized the tremendous potential in targeting PPIs over the last two decades, the development of small molecules, which specifically modulate or disrupt a particular PPI, remains a challenging and risky undertaking.(1
) Commonly, protein-protein interfaces are large and flat, and they lack deep cavities that might serve as good binding sites for small molecules.(5
) Moreover, amino acids at the interfaces of PPIs are flexible and thus pose challenges at conducting computer-guided compound design.(7
Although protein-protein interfaces bury 500–3000 Å2
of total surface area, which exceeds the potential binding area of low-molecular-weight compounds,(10
) Wells and co-workers demonstrated that only a fraction of the amino acid residues at the protein-protein interface contributes to the major portion of the binding free energy.(12
) These key amino acids, defined as recognition patches or hot spots, therefore provide the theoretical and experimental evidence that PPIs can be disrupted or modulated by low-molecular-weight compounds. In the last 15 years, numerous approaches have been developed for the discovery of small molecules modulating or disrupting PPIs. Often, small molecule design is aimed at mimicking a peptide or a protein secondary structure in a truncated form.(15
) Alternatively, fragment-based drug discovery strategies using biomolecular NMR, X-ray crystallography, or surface plasmon resonance (SPR) lead to the identification of fragments with good ligand efficiencies, which are further developed into potent protein-protein interaction modulators (PPIMs). Herein we report the expansion and utilization of kinetic Target-Guided Synthesis (TGS) as a screening platform for the identification of PPIMs.
In the last two decades, several TGS approaches have been described, in which the target biomolecule assembles its inhibitory ligand from a collection of reactive fragments. Depending on the nature of the assembly step, TGS approaches can be classified into (a) dynamic combinatorial chemistry (DCC), (b) reagent-accelerated TGS, and (c) kinetic TGS.(17
) In dynamic combinatorial chemistry, the assembly process is reversible, whereas reagent-accelerated TGS uses building blocks, which combine in an irreversible fashion only in presence of an external reagent or a catalyst upon binding to the biological target. In kinetic TGS, a biological target accelerates the irreversible covalent bond formation only between complementary reacting fragments binding to adjacent binding sites of the target (). Kinetic TGS(16
) and in situ
) have been exclusively applied for the identification of inhibitors of enzymatic targets with well defined binding pockets. In a recent proof-of-concept study with the anti-apoptotic protein Bcl-XL
as the biological target, we demonstrated that kinetic TGS can also be used for the “rediscovery” of a PPIM previously reported by the Abbott Laboratories starting from smaller fragments bearing a thio acid or a sulfonyl azide functional group.(20
) Williams and coworkers described that the amidation reaction between thio acids and sulfonyl azides,(21
) which in the meantime has been named as the sulfo-click reaction,(23
) proceeds in aqueous media.
Figure 1 Kinetic TGS approach targeting PPIs. A) TGS approaches are based on the principle that multidentate interactions between a ligand and a biological target are collectively much stronger than the corresponding monovalent interactions of each of the fragments.( (more ...)
The proteins of the Bcl-2 family have been validated as attractive PPI targets for cancer therapy.(24
) The Bcl-2 family of proteins, which consists of both anti- and pro-apoptotic molecules, plays a pivotal role in the regulation of the intrinsic pathway of apoptosis. The anti-apoptotic Bcl-2 family proteins Bcl-2, Bcl-XL
, and Mcl-1 inhibit the release of certain pro-apoptotic factors from mitochondria. In contrast, pro-apoptotic Bcl-2 family members, which can be further separated into two subgroups, the multidomain BH1–3 proteins (i.e., Bax and Bak) and the BH3-only proteins (e.g., Bad, Bim, and Noxa), induce the release of mitochondrial apoptogenic molecules into the cytosol.(25
) Evidence has been accumulated that the majority of human cancers overexpress the pro-survival Bcl-2 family proteins, which not only contribute to cancer progression by preventing normal cell turnover, but also render cancer cells resistant to current cancer treatments.(27
) Although there is a controversy over how anti-apoptotic Bcl-2 family proteins function,(29
) it is generally accepted that apoptosis is initiated by the binding of pro-apoptotic BH3-only proteins to anti-apoptotic Bcl-2 family molecules in cancer cells. These interactions are mediated by the insertion of the BH3 domain of pro-death proteins into the hydrophobic groove on the surface of anti-apoptotic proteins Bcl-2, Bcl-XL
, or Mcl-1.(31
) Therefore, small molecules that mimic the BH3 domains of pro-apoptotic Bcl-2 family proteins have potential as anti-cancer therapeutics.
Previously, Abbott Laboratories developed acylsulfonamide 1, ABT-737, ABT-263
, and other structurally related acylsulfonamides, which efficiently disrupt Bcl-XL
-Bad interaction ().(33
) Based on these reports, we designed reactive fragments structurally related to ABT-737
), and incubated these as binary fragment mixtures in presence of Bcl-XL
(). Analysis of each incubation sample by liquid chromatography combined with mass spectrometry detection in the selected ion mode (LC/MS-SIM) showed that of all 18 possible products only compound SZ4TA2
, which was developed by Abbott Laboratories, has been detected. In comparison, incubations of fragments in the absence of Bcl-XL
or in presence of Bcl-XL
and various BH3-containing peptides failed to yield detectable amounts of acylsulfonamide products. In addition, IC50
inhibitory constants in the nM range have been determined for SZ4TA2
, while their corresponding thio acid or sulfonyl azide fragments did not show any inhibition up to 100 μM concentrations.
Herein, we successfully employed and validated the sulfo-click kinetic TGS approach as a straightforward yet reliable PPIM screening platform for the identification of Bcl-XL-protein modulators. The design of kinetic TGS incubations with wildtype and mutant Bcl-XL proteins provided an additional layer of confirmatory experiments for the delivery of high-quality PPIMs. Furthermore, experimental evidence has been accumulated indicating that kinetic TGS is a PPIM screening and synthesis method generating only active compounds.