Prolonged and tunable residence time using reversible covalent kinase inhibitors

Abstract

Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here we made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrated biochemical residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.

Figure 1. Reversible covalent BTK inhibitors based on inverted cyanoacrylamides

(a) Cyanoacrylamides, attached via a piperidine linker to a pyrazolopyrimidine scaffold, are capped with alkyl groups of increasing steric demand (1-3). (b) Inhibitors 1-3 have distinct levels of BTK durability in cells following inhibitor washout. Ramos B cells were treated with DMSO or 1-3 (1 μM) for 1 hr, washed 3 times with PBS, and incubated in compound-free media at 37 °C. After 4 or 20 hrs, cells were treated with 1 μM PP-BODIPY (the irreversible probe that labels BTK Cys481) for 1 hr and then lysed and analyzed by in-gel fluorescence. Occupancy was calculated from the normalized in-gel fluorescence intensity divided by the DMSO control value and subtracted from 100% (mean ± SD, n = 3). (c) Co-crystal structure of 3 bound to BTK at 2.2 Å resolution. The covalent bond between Cys481 and Cβ, newly formed CαH, and select hydrogen bonds are indicated. Two of the capping group methyls form hydrophobic contacts with Leu483 and Arg525, while the third is solvent exposed.

Figure 2. Prolonged and tunable residence time of reversible covalent BTK inhibitors

(a) BTK inhibitors with methylpyrrolidine linkers characterized during residence time-based optimization. Compounds contain R (4-5, top) and S (6-9, bottom) linker configurations. Inhibitors 4, 7, 8, 9 contain a cyanoacrylamide electrophile, PP-ir contains an acrylamide electrophile, and PP-ne has no electrophile. (b) Dissociation curves of BTK inhibitors. Shown is the percent BTK with inhibitor bound (% BTK Bound) versus time (hrs) for a 108-hour dissociation experiment. Residence times, τ, were determined as follows: 4: 22 ± 3 hrs; 7: 34 ± 5 hrs; 8: 83 ± 14 hrs; 9: 167 ± 21 hrs; PP-ir: >200 hrs. Data from representative experiments are shown (mean ± SD, n = 2). (c) A 1-hour inhibitor dissociation study. Residence times, τ, were determined as follows: PP-ne: <0.1 hrs; dasatinib: 0.46 ± 0.6 hrs. Data from representative experiments are shown (mean ± SD, n = 2). (d) Prolonged residence time is tunable and not predicted by enzyme IC₅₀ potency. A series of 21 cyanoacrylamide-containing pyrazolopyrimidines with pyrrolidine linkers were characterized for both occupancy after 24 hrs in the biochemical off-rate assay (blue) and for potency in a BTK enzymatic activity assay (green). The occupancy at 24 hrs in the off-rate assay is plotted side-by-side with the IC₅₀ for the same compound (mean ± SD, n ≥ 2). Compounds were sorted left-to-right in descending order of durability, also see Supplementary Table 1.

Figure 3. Long-term cellular durability of reversible covalent BTK inhibitors

(a) Sustained BTK occupancy of compound 9 in cells 4 hrs following inhibitor washout. Ramos B cells were incubated with various concentrations of 9 and either treated with 1 μM PP-BODIPY directly (top) or washed 3 times and returned to culture for 4 hrs prior to adding 1 μM PP-BODIPY (bottom). Lysates were evaluated both for binding of the PP-BODIPY fluorescent probe to BTK (PP-BODIPY) and total BTK by Western blot (Total BTK) (n = 2). See Supplementary Fig. 25 for full gel images. (b) Reversible covalent BTK inhibitors exhibit distinct levels of durability in cells. Ramos B cells were incubated with 1 μM inhibitor, washed 3 times, and returned to culture for 4 hrs prior to addition of 1 μM PP-BODIPY. Lysates were evaluated both for binding of the PP-BODIPY probe and total BTK to calculate % Occupancy (mean ± SD, n ≥ 2) of the inhibitor (See Supplementary Fig. 14). (c) BTK occupancy of reversible covalent inhibitors 4 hrs and 18 hrs following cell washout (mean ± SD, n ≥ 2) evaluated using Alphascreen technology (Supplementary Fig. 15).

Figure 4. Kinase selectivity of inhibitor 9

(a) Kinase selectivity of 9 evaluated at 1 μM and 0.1 μM in the Nanosyn 254 kinase panel. Circles indicate the 6 kinases that showed >90% inhibition at 1 μM. Large circles indicate the 2 kinases (BTK and BMX) that also demonstrated >90% inhibition at 0.1 μM. The figure was reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com). (b) Enzymatic IC50 curves determined using inhibitor 9 toward BTK and other kinases with a homologous cysteine with known tissue-specific homeostatic roles in vivo (EGFR, ERB-B2, ITK, JAK3) (mean ± SD, n ≥ 2). IC50 values and curves toward the other related cysteine-containing kinases are shown in Supplementary Fig. 20. (c) Cellular selectivity of inhibitor 9. The activity of 9 in Ramos B cells using a BTK occupancy assay (see Supplementary Fig. 15) was compared with the activity toward ITK and EGFR in T cell receptor (TCR) and EGFR cellular reporter assays, respectively (mean ± SD, n ≥ 2). In the TCR assay, 9 was studied in a Jurkat T cell line stimulated with anti-CD3 plus anti-CD28 (to activate the T cell receptor pathway) and NFAT reporter expression was measured. In the EGFR assay, 9 was studied in ME-180 cervical carcinoma cells stimulated with EGF (to activate the EGFR receptor pathway) and AP-1 reporter expression was measured.

Figure 5. Extended pharmacodynamic effect of an orally bioavailable BTK inhibitor

(a) PK/PD relationship for 9 in rats dosed orally at 40 mg/kg. The percent BTK occupancy (in blue) and the concentration of 9 in plasma (in green) at each time point following dosing is shown (mean ± SD, n = 3). The potency of 9 in the human whole blood CD69 expression assay was 146 ± 10 ng/ml. (b) The gel-based data used to calculate the BTK occupancy of 9. Occupancy was determined with in-gel fluorescence using rat PBMC collected from 3 separate animals at each time point. The % BTK occupancy was determined from the percentage block of BODIPY probe compared to vehicle treated animals (ctrl) with all samples normalized for the total amount of BTK as evaluated by Western blotting (n = 2). See Supplementary Fig. 25 for full gel images.

Figure 6. Inverted cyanoacrylamide FGFR inhibitors with prolonged, tunable residence time

(a) Model of the kinase ATP binding site demonstrating the disparate locations of cysteine residues targeted by cyanoacrylamide-based RSK2, BTK, and FGFR inhibitors. Structural elements are colored as follows: green:glycine rich loop; purple: αC-helix; cyan:DFG loop; yellow:hinge region; red: phosphate binding region. (b) Inverted cyanoacrylamides linked to a pyridopyrimidinone scaffold were synthesized with a variety of different capping groups. (c) Dissociation curves of FGFR inhibitors. Shown is the percent FGFR1 with inhibitor bound (% FGFR1 Bound) versus time (hrs) for a 24-hour dissociation experiment. Residence times, τ, were determined as follows: 28: >150 hrs; 29: 110 ± 41 hrs; 30: 18 ± 5 hrs; BGJ398: 1.9 ± 0.4 hrs. Data from representative experiments are shown (mean ± SD, n = 2). (d) Prolonged and tunable residence time of inverted cyanoacrylamide FGFR inhibitors. FGFR inhibitors 28 through 38 containing a variety of electrophile capping groups were characterized (See compounds and durability data in Supplementary Table 4). Inhibitors were found to display a continuum of residence times as evaluated by the % FGFR1 occupancy 24 hours after addition of a fluorescent competitive tracer (orange bars) (mean ± SD, n ≥ 2). Residence time was not predicted by potency in a FGFR1 enzymatic inhibition assay (purple bars) since IC50 potency for all compounds clustered in the range between 0.5 ± 0.05 nM and 6.0 ± 0.3 nM.