Graduation Year


Document Type




Degree Granting Department


Major Professor

Roman Manetsch


Acylsulfonamides, Amidation, Amidomethylarenes, Fragment-based lead discovery, Protein-protein interaction modulators, Sulfo-click chemistry


Kinetic target-guided synthesis (TGS) and in situ click chemistry are among unconventional discovery strategies having the potential to streamline the development of protein-protein interaction modulators (PPIMs). In kinetic TGS and in situ click chemistry, the target is directly involved in the assembly of its own potent, bidentate ligand from a pool of reactive fragments. Herein, we report the use and validation of kinetic TGS based on the sulfo-click reaction between thio acids and sulfonyl azides as a screening and synthesis platform for the identification of high-quality PPIMs. Starting from a randomly designed library consisting of nine thio acids and nine sulfonyl azides leading to eighty one potential acylsulfonamides, the target protein, Bcl-XL selectively assembled four PPIMs, acylsulfonamides SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5, which have been shown to modulate Bcl-XL/BH3 interactions. To further investigate the Bcl-XL templation effect, control experiments were carried out using two mutants of Bcl-XL. In one mutant, phenylalanine Phe131 and aspartic acid Asp133, which are critical for the BH3 domain binding, have been substituted by alanines, while arginine Arg139, a residue identified to play a crucial role in the binding of ABT-737, a BH3 mimetic, has been replaced by an alanine in the other mutant. Incubation of these mutants with the reactive fragments and subsequent LC/MS-SIM analysis confirmed that these building block combinations yield the corresponding acylsulfonamides at the BH3 binding site, the actual "hot spot" of Bcl-XL. These results validate kinetic TGS using the sulfo-click reaction as a valuable tool for the straightforward identification of high-quality PPIMs.

Protein-protein interactions of the Bcl-2 family have been extensively

investigated and the anti-apoptotic proteins (Bcl-2, Bcl-XL, and Mcl-1) have been validated as crucial targets for the discovery of potential anti-cancer agents. At the outset, Bcl-2 and Bcl-XL were considered to play an important role in the regulation of apoptosis. Accordingly, several small molecule inhibitors targeting Bcl-2 and/or Bcl-XL proteins were primarily designed. A series of acylsulfonamides targeting these proteins were reported by Abbott laboratories, ABT-737 and ABT-263 being the most potent candidates. Remarkably, these molecules were found to exhibit weaker binding affinities against Mcl-1, another anti-apoptotic protein. Further experimental evidence suggests that, inhibitors targeting Mcl-1 selectively or in combination with other anti-apoptotic proteins would lead to desired therapeutic effect. As a result, numerous small molecules displaying activity against Mcl-1 have been identified so far. Specifically, acylsulfonamides derived from structure activity relationship by interligand nuclear overhauser effect (SAR by ILOEs), a fragment-based approach, have been recently reported with binding affinities in the nanomolar range. In the meantime, we have reported that the kinetic TGS approach can also be applied to identify acylsulfonamides as PPIMs targeting Bcl-XL. Taken together, structurally novel acylsulfonamides can be potentially discovered as Mcl-1 inhibitors using the kinetic TGS approach. Thus, a library of thirty one sulfonyl azides and ten thio acids providing three hundred and ten potential products was screened against Mcl-1 and the kinetic TGS hits were identified. Subsequently, control experiments involving Bim BH3 peptide were conducted to confirm that the fragments are assembled at the binding site of the protein. The kinetic TGS hits were then synthesized and subjected to the fluorescence polarization assay. Gratifyingly, activities in single digit micromolar range were detected, demonstrating that the sulfo-click kinetic TGS approach can also be used for screening and identification of acylsulfonamides as PPIMs targeting Mcl-1.

The amide bond serves as one of nature's most fundamental functional group and is observed in a large number of organic and biological molecules. Traditionally, the amide functionality is introduced in a molecule through coupling of an amine and an activated carboxylic acid. Recently, various alternative methods have been reported wherein, the aldehydes or alcohols are oxidized using transition metal catalysts and are treated with amines to transform into the corresponding amides. These transformations however, require specially designed catalysts, long reaction times and high temperatures. We herein describe a practical and efficient amidation reaction involving aromatic aldehydes and various azides under mild basic conditions. A broad spectrum of functional groups was tolerated, demonstrating the scope of the reaction. Consequently, the amides were synthesized in moderate to excellent yields, presenting an attractive alternative to the currently available synthetic methods.