Functionalized Scout Fragments for Site-Specific Covalent Ligand Discovery and Optimization

Abstract

Covalent ligands are a versatile class of chemical probes and drugs that can target noncanonical sites on proteins and display differentiated pharmacodynamic properties. Chemical proteomic methods have been introduced that leverage electrophilic fragments to globally profile the covalent ligandability of nucleophilic residues, such as cysteine and lysine, in native biological systems. Further optimization of these initial ligandability events without resorting to the time-consuming process of individualized protein purification and functional assay development, however, presents a persistent technical challenge. Here, we show that broadly reactive electrophilic fragments, or "scouts", can be converted into site-specific target engagement probes for screening small molecules against a wide array of proteins in convenient gel-and ELISA-based assay formats. We use these assays to expediently optimize a weak potency fragment hit into a sub-μM inhibitor that selectively engages an active-site cysteine in the retinaldehyde reductase AKR1B10. Our findings provide a road map to optimize covalent fragments into more advanced chemical probes without requiring protein purification or structural analysis.

Conditional covalent lethality driven by oncometabolite accumulation

“…Related to this issue is the fact that almost all chemoproteomic methods, including the IA-alkyne based method applied here, sample only a subset of the cellular cysteineome. Interrogating additional time points and concentrations using alternative enrichment agents, [36][37][38] including 'scout probes' capable of in cell cysteine reactivity profiling, 16,17,39 may yield additional functional MP-1 targets. A second takeaway is that while TRMT1 and m2,2G levels do not appear to be modulated by loss of FH, the reactivity of TRMT1's C620/621 residue is reduced upon fumarate accumulation.…”

Reactive chemistry for covalent probe and therapeutic development

“…Chemical proteomic studies, however, have shown that even highly optimized acrylamide kinase drugs exhibit off-target activity when evaluated in live cells and animal models [27,29]. Medicinal chemistry optimization combined with comprehensive selectivity profiling (e.g., using ABPP; Box 1 and Figure 2) is a strategy for mitigating off-target activity of acrylamide compounds [30]. The development of electrophiles with tunable cysteine reactivity offers additional opportunities for achieving selectivity [31].…”

Perspective on the Structural Basis for Human Aldo-Keto Reductase 1B10 Inhibition

“…In the first work [37], they used a broadly reactive iodoacetamide alkyne (IA-alkyne, Figure 1) in lung cancer cell lines and identified three liganded proteins exclusive to KEAP1-mutant cells (KEAP1 is a negative regulator of the transcription factor NRF2, which in cancer cells induces expression of metabolic enzymes such as AKR1B10 to restore redox homeostasis). In the second study [38], they have developed a second type of broadly reactive (but less unspecific) electrophilic fragment ("scout" fragments, Figure 1) with the same purpose of mapping Cys ligandability. AKR1B10 is used as a proof-of-concept, and again Cys299 has been identified as a highly reactive Cys with the scout fragments.…”

“…A common feature of both types of covalent inhibitors is that their discovery involved screening with cell lysates, not living cells [28,38]. Surprisingly, the most potent lead, VC59, did not bind to AKR1B10 in living lung cancer cells [38]. The researchers found out that, in cell lysates, increasing concentrations of NADPH prevented reactivity of Cys299.…”