Reactive chemistry for covalent probe and therapeutic development

Abstract

Bioactive small molecules that form covalent bonds with a target protein are important tools for basic research and can be highly effective drugs. This review highlights reactive groups found in a collection of thiophilic and oxophilic drugs that mediate pharmacological activity through a covalent mechanism of action (MOA). We describe the application of advanced proteomic and bioanalytical methodologies for assessing selectivity of these covalent agents to guide and inspire the search for additional electrophiles suitable for covalent probe and therapeutic development. While the emphasis is on chemistry for modifying catalytic serine, threonine or cysteine residues, we devote a substantial fraction of the review to a collection of exploratory reactive groups of understudied residues on proteins.

Keywords: activity-based protein profiling; chemoproteomics; drug discovery; electrophile; targeted covalent inhibitor.

Figure 1.. Thiophilic drugs and (seleno)cysteine reactive electrophiles.

(A) Progression of SAR studies, leading to sotorasib and the co-crystal structure of the sotorasib-Cys12 adduct on KRAS^G12C (PDB ID: 6OIM). (B) Chemical structure of ibrutinib (Imbruvica^®) and the co-crystal structure showing the ibrutinib-Cys481 adduct in BTK (PDB ID: 5P9J). (C) BODIPY (PCI-33380) and alkynyl (Probe 4) derivatives of ibrutinib as probes for ABPP (See Box 1). (D) Masked nitrile oxide electrophile reported by Schreiber et al. and the proposed MOA for selective labeling of selenocysteine.

Figure 2.. Schematic of activity-based protein profiling.

(ABPP) using gel electrophoresis (A) or LC-MS/MS detection (B). (C) Structures of reactive groups, multiplexing strategies, and types of ABPs used for ABPP analysis. SILAC: stable-isotope labeling using amino acids in cell culture; CuAAC: copper-catalyzed azide-alkyne [3+2]-cycloaddition; LC-MS/MS: liquid chromatography-tandem mass spectrometry. Samples analyzed are typically derived from studies of compound/probe treatments in lysates, cells, or animals. Clinical samples can also be subjected to this approach. See Box 1 for the supporting text to introduce ABPP methodology.

Figure 3.. Oxophilic drugs and mechanisms of reactivity.

(A) General reaction between phenylboronic acid and water. (B) Phenylboronic acid derivative of darunavir and a schematic representation of the low-barrier hydrogen bond with Asp30 in HIV-1 protease. (C) FDA-approved drugs containing boron. (D) Co-crystal structure of ixazomib-Thr1 adduct in human 20S proteasome (PDB ID: 5LF7). (E) Co-crystal structure of tavaborole-cAMP adduct in leucyl tRNA (tRNA^Leu) synthetase (PDB ID: 2V0C). (F) Co-crystal structure of crisaborole derivative an2898 adducted to an activated water molecule (H₂O*), which is more nucleophilic due to coordination of the oxygen in water with Mg²⁺ and Zn²⁺, in phosphodiesterase 4 (PDE4; PDB ID: 3O0J). Purple sphere: Zn²⁺; Red sphere: H₂O; Yellow sphere: Mg²⁺. (G) Skeletal drawing of orlistat and the suggested mechanism for covalent inhibition of fatty acid synthase (FASN).

Figure 4.. Oxophilic reactive groups in drugs and chemical probes.

(A) Progression of the MAGL inhibitor JZL-184 to HFIP-carbamates KML-29 and ABX-1431; the latter analog is currently in clinical evaluation for treatment of neurological disorders. (B) Left panel: general reaction scheme between a 1,2,3-triazole-urea and a nucleophilic alcohol (found on serine side chain group) that forms a Michaelis-Menten complex followed by release of the triazole to produce a carbamate adduct group (AG). Right panel: trend for tuning the reactivity of heterocyclic-ureas for developing serine hydrolase inhibitors.

Figure 5.. Representative examples of pharmacologically relevant sulfur(VI)-electrophiles.

(A) Sulfur-fluoride exchange (SuFEx) chemistry including sulfonyl fluorides, aryl fluorosulfates, and sulfonimidoyl fluorides for synthetic and medicinal chemistry. (B) Sulfonyl fluoride XO44 as a broadly reactive probe for kinome ABPP studies. (C) Scheme showing selective reactivity against orthogonal residues on DcpS by deploying a fluorosulfate (FS-p1) or sulfonyl fluoride (SF-p1) electrophile. (D) General reaction scheme between a sulfonyl-triazole and a tyrosine, i.e., sulfur(VI)-triazole exchange (SuTEx), the reactivity of which can be tuned by adjusting the electronics on the adduct group and/or the leaving group (LG). (E) Structure of TH211 and KY-26, both of which are general SuTEx probes of the kinome, as well as HHS-0701, an inhibitor of prostaglandin reductase 2 (PTGR2).