The chemical reaction employed by KTGS is key to its remarkable success. The sulfo-click amidation has been vital in developing KTGS for targeting protein-protein interactions (PPIs). Many biological processes rely on the interaction between different proteins, and therefore, PPIs represent a crucial and extensive category of potential targets for developing novel therapies [15, 16]. The first instance of KTGS being used to target PPIs was reported by Manetsch’s group [17], which identified PPI modulators through the sulfo-click amidation. A range of thioacids and sulfonyl azides were synthesized and employed in the KTGS strategy to identify potent inhibitors of B cell lymphoma-extra large (Bcl-xL). Bcl-xL is a crucial member of the Bcl-2 family that plays a critical role in regulating the intrinsic pathway of apoptosis. In this study, thioacids and sulfonyl azides were incubated as binary mixtures (18 different incubations, each incubation containing one thioacid and one sulfonyl azide) with and without Bcl-xL, and the formation of acylsulfonamide products was analyzed by LC-MS/MS. Only one of the 18 possible combinations displayed the desired Bcl-xL-templated effect (Scheme 2) [17]. The hit compound SZ4TA2 was then synthesized chemically and tested for its ability to inhibit the interaction between Bcl-xL and Bak using a fluorescence polarization (FP) competition assay. The hit compound SZ4TA2 displayed a remarkable ability to inhibit the interaction of Bcl-xL and Bak with an IC50 of 78.8 nM.

Bcl-xL-templated binary fragment KTGS utilizing 6 sulfonyl azides (each at 20 µM) and 3 thioacids (each at 20 µM) potentially leading to 18 different acylsulfonamide products

The study on the Bcl-xL-templated sulfo-click amidation was extended further using a more extensive fragment library to identify novel PPI inhibitors via the binary fragment KTGS strategy. Four of 81 possible combinations showed the templated effect, identifying three new hits, namely SZ7TA2, SZ9TA1, and SZ9TA5, and the previously identified hit SZ4TA2 (Scheme 3) [18]. All four hits were found to exhibit PPI modulatory activity when analyzed for their ability to modulate the PPI using a fluorescence-based competitive binding assay.

Bcl-xL-templated binary fragment KTGS utilizing 9 sulfonyl azides (each at 20 µM) and 9 thioacids (each at 20 µM) leading to 81 different acylsulfonamide products

Although the sulfo-click amidation has been deemed reliable in various applications, it has not been widely used due to limitations regarding the preparation and handling of thioacids. Manetsch’s group successfully employed a one-pot deprotection/sulfo-click amidation approach in KTGS, overcoming this challenge. In this deprotection/sulfo-click amidation approach, 9-fluorenylmethyl (Fm) thioesters were quickly deprotected into thioacids using 5% piperidine in DMF and then reacted with sulfonylazides to produce N-acylsulfonamides (Scheme 4) [19]. The method has proven to be highly effective and has the potential to revolutionize the use of sulfo-click amidation in various applications.

Sulfo-click amidation via in situ generated thioacid and its application in KTGS

While the binary KTGS approach using sulfo-click amidation has yielded promising results, the method requires LC-MS analysis of a large number of incubations, limiting the throughput of the KTGS screening platforms. As a result, the multi-fragment KTGS approach has been developed to improve the efficiency of KTGS in accessing a more extensive chemical space. The multi-fragment KTGS approach holds the potential to increase the screening platform’s throughput dramatically. The developed multi-fragment KTGS approach allowed the Manetsch laboratory to screen 1710 possible fragment combinations in 18 wells of a 96-well plate (190 fragment combinations in a single well; nine wells with the protein target and another nine wells without the protein target), utilizing an 83-member fragment library (Fig. 2) with and without myeloid cell leukemia-1 (Mcl-1) and analyzed by LC-MS/MS. Mcl-1, a protein belonging to the Bcl-2 family, is known for its anti-apoptotic properties. Its role in cancer cell survival and resistance to chemotherapy has made it a promising target for cancer therapy. Among the 51 total hits (Scheme 5), our findings identified 24 Mcl-1 inhibitors with single-digit micromolar IC50 values (Table 1) [20], as confirmed by fluorescence polarization (FP) studies. These results demonstrate that the multi-fragment sulfo-click KTGS approach is highly effective at developing hit compounds targeting Mcl-1. This finding is a breakthrough in KTGS screening as it reports the largest number of unique combinations per well ever recorded, opening up new possibilities for research in the fragment-based lead discovery field.

Chemical structures of the “in-house” library of 83 fragments (38 sulfonyl azides × 45 in situ prepared thioacids = 1710 possible combinations)

Mcl-1-templated multi-fragment KTGS utilizing 38 sulfonyl azides (each at 20 µM) and 45 in situ formed thioacids (each at 20 µM) leading to 1710 possible combinations

Next, the above-developed multi-fragment KTGS screening method was employed to identify inhibitors of pathogenic free-living amoebae (pFLA) glucokinase (Glck) enzymes. pFLA is notorious for inducing severe and potentially life-threatening infections of the central nervous system. Therefore, it is essential to investigate and explore novel strategies that can effectively prevent and treat these lethal infections. From an in-house library of 83 fragments (Fig. 2), 157 hit compounds were identified via the multi-fragment KTGS approach (Scheme 6). Among the total 157 hits, twelve inhibitors were identified against three pFLA Glck enzymes - Naegleria fowleri (Nf) Glck, Balamuthia mandrillaris (Bm) Glck, and Acanthamoeba castellanii (Ac) Glck (Table 2) [21]. The fragments were not selected based on predetermined binding potentials, indicating that KTGS, in combination with randomly designed fragments, can effectively screen chemical space and discover new compounds with medicinal chemistry potential. This demonstrates the usefulness of the multi-fragment KTGS screening in drug discovery research.

NfGlck-templated multi-fragment KTGS utilizing 38 sulfonyl azides (each at 20 µM) and 45 in situ formed thioacids (each at 20 µM) leading to 1710 possible combinations