Sulfamate Acetamides as Self-Immolative Electrophiles for Covalent Ligand-Directed Release Chemistry

Abstract

Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA-approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry with tunable reactivity. In the context of the BTK inhibitor ibrutinib, sulfamate analogues showed low reactivity with comparable potency in protein labeling, in vitro, and cellular kinase activity assays and were effective in a mouse model of CLL. In a second example, we converted a chloroacetamide Pin1 inhibitor to a potent and selective sulfamate acetamide with improved buffer stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of "turn-on" probes as well as for traceless ligand-directed site-specific labeling of proteins. Taken together, this chemistry represents a promising addition to the list of electrophiles suitable for in vivo covalent targeting.

Figure 1

Sulfamate acetamides as electrophiles for targeted covalent inhibitors and CoLDR chemistry: (A) Reactivity pattern of α-substituted acetamides. (B) Schematic representation of the reaction of a target cysteine with α-sulfamate acetamides through CoLDR chemistry.

Figure 2

α-Sulfamate acetamides can show up to two orders of magnitude less reactivity towards GSH than chloroacetamide. (A) Chemical structures of model α-sulfamate/sulfonate/sulfone acetamides. (B) Half-life (t_1/2) of the model compounds (1a–1j) assessed by GSH consumption assay via LC/MS (Figure S3B). (C). In situ proteomic labeling with alkyne probes (2a–2c). Mino cells were treated for 2 h with either DMSO or 2a–2c, then lysed, reacted with TAMRA-azide using CuAAC, and imaged via in-gel fluorescence (532 nm). Bands that are selectively detected only by compound 2a are indicated by asterisks.

Figure 3

Ibrutinib sulfamates as potent BTK inhibitors: (A) Chemical structures of ibrutinib, 3a–3g. (B) Deconvoluted LC/MS spectrum of BTK (2 μM) incubated with 3c (2 μM) at pH 8, 25 °C, 30 min. The adduct mass corresponds to a labeling event in which methyl sulfamic acid was released, validating the proposed mechanism. (C) % of labeling of BTK (2 μM) with the probes (3a–3e; 2 μM) at 10 min (blue bar) and 30 min (green bar) in 20 mM Tris buffer at pH 8, 25 °C. (D) In vitro kinase activity assay (0.5 nM BTK, 5 μM ATP) for 3a–3g (see Figure S11 for IC₅₀ values). (E) Correlation of GSH half-life (t_1/2) of ibrutinib sulfamates with measured IC₅₀s in a kinase inhibition assay.

Figure 4

Ibrutinib sulfamate acetamide analogues are highly potent in cells and in vivo. (A) Dose-dependent BTK activity assay in Mino cells as measured by autophosphorylation of BTK. The cells were incubated for 2 h with either 0.1% DMSO, various concentrations of ibrutinib, or 3a–3d. The cells were activated with anti-IgM, and BTK autophosphorylation was quantified by Western blot and normalized with respect to β-actin. IC₅₀s were calculated by fitting the data to a dose–response curve using Prism software. (B) Dose-dependent inhibition of B-cell response (as measured by CD86 expression) after anti-IgM-induced activation and treatment with ibrutinib analogues (3a–3d) for 24 h (n = 3; error bars indicate standard deviation). (C) Dose-dependent inhibition of pBTK and its downstream pathways (pPLCγ2 , pAkt, and pERK) by ibrutinib derivatives (3a, 3c, 3d, and 3e) in CLL patient samples. CLL cells (20 × 10/mL) were incubated with ibrutinib or ibrutinib-based compounds at the indicated doses at 37 °C. DMSO-treated cells served as controls. After 2 h of incubation, the cells were either stimulated with goat F(ab′)2 anti-human IgM (10 μg/mL) for 15 min or left untreated. Proteins were then extracted and subjected to Western blot analysis. (D) Schematic representation of the in vivo mice experiment. Cells isolated from old TCL1 mice spleens, with a malignant cell population higher than 60%, were injected into the tail vein of 6 week old recipient mice. The mice were given a solution containing sulfamate 3c (0.16 mg/mL in 1% cyclodextrin water) ad libitum in drinking water. Progression of the disease was followed in the peripheral blood (PB) by using flow cytometry for quantification of the IgM+/CD5+ population (created with BioRender.com). (E) IgM+/CD5+ cell population is significantly lowered in 3c-treated mice (n = 5) compared to untreated (n = 3). **p = 0.002 for days 7 and 15 (single-tailed Student’s t test) (F) BTK engagement of compound 3c in vivo. Dissected spleens were extracted with RIPA buffer and incubated with an ibrutinib alkyne analogue (probe-4) for 1 h followed by CuAAC reaction with TAMRA-azide in lysate before imaging. (G) IsoDTB-ABPP experiment with ibrutinib and sulfamate 3c. Mino cells were treated with 1 μM of either ibrutinib or 3c for 2 h, followed by incubation of iodoacetamide alkyne and CuAAC click reaction with heavy/light isoDTB tags. The labeled peptides were pulled down with streptavidin beads and quantified via LC/MS/MS (Figure S21; n = 4). Proteins in the box have a heavy-to-light (H/L) ratio of ≥ 2. Only peptides detected in at least three out of four repetitions are presented (Dataset S1). (H) Selectivity of ibrutinib, 3c, and 3d quantified via a competitive pull-down proteomics experiment. Mino cells are treated with 1 μM compound for 1 h and 10 μM ibrutinib alkyne for an additional 1 h (n = 4). Proteins were quantified using label-free quantification. Proteins in the box show a significant change (Fold change > 4; p < 0.05).

Figure 5

Sulfopin sulfamate acetamides as potent and selective Pin1 inhibitors. (A) Chemical structures of the sulfopin analogs (4a–4g). (B) Deconvoluted LC/MS spectrum for Pin1 (2 μM) incubated with 4g (2 μM) at pH 7.5, 25 °C, 1 h. The adduct mass corresponds to a labeling event in which the sulfamate group was released. (C) Percent of Pin1 labeling (2 μM) with the probes (4a–4g; 2 μM; y axis) compared to their intrinsic thiol reactivity as assessed by their rate of reaction in a DTNB assay (x axis). (D) Cellular engagement of the sulfopin sulfamates. OCI-AML2 cells were treated with DMSO, sulfopin, or sulfamates (4c, 4d, and 4 g) at 0.5 and 2.5 μM concentration for 4 h. Lysates were then incubated with a sulfopin DTB probe (1 μM; Figure S29) pulled down using streptavidin beads before running a Western blot against Pin1. (E) IsoDTB ABPP experiment with sulfopin and sulfamate compound 4d. PATU-8988 T cells were treated with 2.5 μM compound for 4 h followed by incubation of iodoacetamide alkyne and CuAAC click reaction with heavy/light azides containing DTB tags. The labeled peptides were pulled down with streptavidin beads and analyzed by LC/MS/MS (similar protocol to BTK; see Figure S21; n = 4). Proteins in the box have a heavy to light (H/L) ratio of ≥ 2. Only peptides detected in three out of four repetitions are presented.

Figure 6

Sulfamate chemistry-based covalent ligand-directed release (CoLDR) probes. (A) Chemical structure of ibrutinib-based “turn-on” releasing probe (3h). (B) Time-dependent increase of fluorescence intensity (representing the release of the coumarin moiety) measured at Ex/Em = 385/435 nm (n = 3). The compound in and of itself (2 μM) is not fluorescent (green). Upon mixing of probe and target (2 μM; blue), we see an increase in fluorescence. Pre-incubation of the protein with ibrutinib prevents the fluorescence (orange). (C) Deconvoluted LC/MS spectra for BTK incubated with 3h at the end of the fluorescence measurement. The adduct mass corresponds to a labeling event in which the coumarin sulfamate (indicated as X) moiety was released, validating the proposed mechanism. (D) Schematic representation of the tagging of proteins with the release of ligand. The target cysteine reaction at the electrophilic sulfamate center is followed by the concomitant release of the ligand through CoLDR chemistry. (E) Chemical structures of ibrutinib-directed sulfamates with methyl and alkyne tag. (F) Deconvoluted LC/MS spectrum shows the labeling of alkyne probe (3j) and demonstrates Ibr-H leaving (2 μM BTK, 2 μM 3j, pH 8.0, 25 °C, 10 min). (G) Cellular labeling profile of 3j (100 nM) after 2 h of incubation with Mino cells. The samples were further reacted with TAMRA-azide in lysate before imaging. An arrow indicates BTK’s MW. Upon competition with ibrutinib (preincubated for 30 min; 1 μM), BTK labeling by 3j is lost. (H) BTK activity assay: Mino cells were incubated for 2 h with either DMSO or 1 μM 3j and then incubated for 45 min with ibrutinib (100 nM). The cells were washed before induction of BTK activity by anti-IgM. The CoLDR probe was able to rescue BTK activity from inhibition by ibrutinib.