BTK (DC50 = 1 nM); E3 ligase; PROTAC degrader

MT-802 degrades BTK with a DC50 of 9.1 nM, reaching a maximum degradation threshold of 250 nM[1].

---

MT-802 Is a Potent and Rapid Degrader of BTK [1]
In our initial characterization experiments, we showed that MT-802 degrades BTK with a DC50 of 9.1 nM, with maximal degradation being observed by 250 nM. Because PROTACs work via a ternary complex-driven mechanism, a common observation for many PROTACs is the “hook effect”, whereby binary species (BTK–PROTAC and PROTAC–cereblon) can predominate over the productive ternary complex at sufficiently high PROTAC concentrations, thereby resulting in a reduced level of degradation. However, we did not observe any significant rebound in BTK levels (i.e., a “hook”) in cells treated with ≤2.5 μM MT-802 (Figure S2). PROTACs inducing ternary complexes with significant positive cooperativity would be expected to have an expansion in the concentration range of their maximal effect due to the diminished presence of the unproductive binary complexes. The lack of an observable hook effect suggests that MT-802 induces a high-affinity ternary complex with significant positive cooperativity. We next synthesized SJF-6625, an inactive version of MT-802 that is incapable of binding to cereblon because of methylation of the glutarimide ring of the pomalidomide moiety (Figure 1A). As expected, neither ibrutinib nor SJF-6625 was able to induce degradation of BTK (Figure 1B), demonstrating that binding to cereblon is required for MT-802’s mechanism of action. [1]
Because MT-802 could elicit complete BTK knockdown at 250 nM, we decided to employ this concentration of compound in our follow-up characterization experiments. At this concentration, we showed that MT-802 fully degrades BTK as early as 4 h, with half of the total BTK degraded after approximately 50 min (Figure 1C and Figure S3A). Pretreatment with epoxomicin, a proteasome inhibitor, followed by treatment with MT-802 did not result in BTK degradation, indicating that proteasome function is required for BTK knockdown. The same was observed after pretreatment with MLN-4924, an inhibitor of the NEDD8-activating enzyme that neddylates and thereby activates many cullin-RING ligases, including the cullin-4A-based cereblon complex. The necessity for direct binding to both BTK and cereblon was shown by pretreating cells with an excess of either ibrutinib or pomalidomide, each of which rescued BTK levels in response to MT-802 (Figure 1D and Figure S3B). These assays demonstrate that MT-802 directly engages BTK and cereblon to engender knockdown in a proteasome-dependent manner.

---

Enhanced Kinase Selectivity by MT-802 over Ibrutinib [1]
Having demonstrated that MT-802 is capable of potent BTK degradation and established the bona fides of its mechanism, we wanted to assess the specificity of MT-802’s binding within the kinome. In general, our group and others have shown that the potency of in vitro kinase binding decreases when the linker and E3-targeting moiety are appended to the parent warhead. It is known that ibrutinib shows off-target inhibition of other kinases, particularly those with cysteines homologous to C481 in BTK. Because MT-802 lacks the acrylamide moiety that binds C481, we reasoned that our PROTAC may bind fewer off-target kinases than ibrutinib does. If confirmed, this finding would be relevant to efforts to develop more specific BTK-targeting agents that are free of the negative side effects of ibrutinib, which include adverse cardiac, gastrointestinal, and skin events. To address this, we utilized KINOMEscan, the high-throughput, competition-based binding assay service provided by DiscoverX. This assay reports binding as a “percentage of control”, where lower values represent higher levels of kinase binding. Using this assay, we screened ibrutinib and MT-802 in parallel at 1.0 μM against a panel of 468 human kinases (Figure S4A,B). Previously assembled data sets on ibrutinib’s kinome-wide inhibition showed reasonable correlation with our own data set (Figure S4C). As expected, BTK was among the most maximally bound kinases by both compounds (0.0 and 0.25% of control for ibrutinib and MT-802, respectively). The only other kinase in the Tec family that was thoroughly bound by both ibrutinib and MT-802 was TEC (1.9 and 3.6% of control, respectively) (Figure 2A). BMX showed weaker binding to both ibrutinib and MT-802, and while TXK could be strongly bound by ibrutinib, MT-802 showed weaker engagement (3.8 and 63% of control, respectively) (Table S2). Of note, MT-802 also bound equally well to ERBB3 (0.0% of control for both MT-802 and ibrutinib) in the KINOMEscan data set, but this binding did not lead to ERBB3 degradation when tested in OVCAR8 cells (Figure S5). This example underscores our previous observation that effective target engagement does not always correlate with target degradation and that other factors such as ternary complex affinity and lysine accessibility may also be relevant.

---

MT-802 Degrades Wild-Type and C481S Mutant BTK [1]
While we did not observe significant degradation of ERBB3, which possesses a serine at the position homologous to cysteine 481, we were encouraged to see that MT-802 nonetheless retained binding to a kinase with this substitution. This suggested that MT-802 has potential to retain interaction with the C481S mutant of BTK, which has been reported in CLL patients exhibiting relapse to ibrutinib therapy. Relapse is proposed to occur because of the loss of the covalent acceptor site, which makes the kinase sensitive only to the reversible inhibition provided by ibrutinib, which is at least 40-fold less potent in vitro (Figure 3A). Having already observed potent degradation of wild-type BTK, we proposed that the loss of ibrutinib’s covalent acceptor position would be inconsequential for MT-802’s ability to degrade BTK because of the PROTAC’s need for only a transient association to induce ubiquitination and knockdown. The C481S resistant context, therefore, would serve as an example in which the event-driven paradigm of PROTACs can perhaps evade a resistance mechanism arising in response to the occupancy paradigm of inhibitors.

---

MT-802 Outperforms Ibrutinib in C481S Primary CLL Patient Samples [1]
To compare the PROTAC to other BTK-targeting moieties, we studied a range of doses and exposure times of patient cells to MT-802. Treatment-naïve B-lymphocytes were isolated from the blood of patients presenting with CLL. Consistent with our experiments with immortalized cell lines, we observed potent knockdown of BTK in the B-lymphocytes of all patients tested (Figure S9). To examine the trends of BTK degradation over multiple doses and time points, a mixed effects model was applied to the log-transformed data to estimate differences relative to vehicle or no treatment. p values for comparisons have been adjusted using the Dunnett–Hsu method (for comparisons against vehicle control). Our dose–response study shows statistically significant degradation is observed as low as 0.1 μM PROTAC (Figure 4A). Time course experiments showed that maximal degradation was observed between 4 and 12 h, and the first signs of statistically significant degradation were seen at just 2 h of treatment (Figure 4B). However, employing a rationale similar to that of our experiments in NAMALWA, we chose 1.0 μM PROTAC for follow-up experiments because of its ability to induce complete knockdown of BTK as early as 12 h. Taken together, these experiments confirm the ability of the PROTAC to degrade BTK in isolated patient B-cells.

KINOMEscan Profiling and in Vitro Kinase Competitive Binding Assays [1]
Ibrutinib and MT-802 were submitted as 1 mM stock solutions in neat DMSO to DiscoverX for the scanMAX service, which screens compounds for binding against a panel of 468 kinases. Both compounds were screened at an assay concentration of 1.0 μM. The assay principle and design have been previously reported. For KdELECT experiments, DiscoverX utilizes the same platform that is employed for the scanMAX service, only expanded across 11 concentration points in duplicate. For KdELECT experiments, the highest concentration of the compound employed was 3.0 μM.
In vitro competition assays for measuring binding to wild-type and C481S BTK were performed by Reaction Biology Corp. IC50 values were determined by fitting a 10-point dose–response curve generated from successive 3-fold dilutions starting at either 10 μM (MT-802) or 1 μM (ibrutinib and compound 1). This assay measures the ability of kinase to directly phosphorylate the substrate in the presence of the compound. All competitive binding curves were generated in the presence of 10 μM ATP.

Cell Treatment and Immunoblotting [1]
For immortalized cell lines, 2 × 10~6 cells per PROTAC treatment condition were collected and washed once with ice-cold PBS, followed by lysis in buffer containing 20 mM Tris (pH 8.0), 0.25% sodium deoxycholate, and 1% Triton X-100, supplemented with protease inhibitors and phosphatase inhibitors (10 mM NaF, 2 mM Na3VO4, 10 mM β-glycerophosphate, and 10 mM sodium pyrophosphate). Lysates were centrifuged at 15000g for 10 min at 4 °C and the supernatant was quantified for total protein content using the Pierce BCA Protein Assay. Thirty micrograms of protein was loaded onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, transferred onto nitrocellulose membranes, and probed with the specified primary antibodies overnight while being rocked at 4 °C in 1× TBS-T (TBS-Tween) containing 5% nonfat milk. HRP-conjugated secondary antibodies were incubated with the membranes for 1 h at room temperature at 1:10000 dilutions in 5% nonfat milk in 1× TBS-T. Imaging was performed using the ECL Prime chemiluminescent Western blot detection reagents followed by visualization with the Bio-Rad ChemiDoc imaging instrument. All Western blots were subsequently processed and quantified using the accompanying Bio-Rad Image Lab software. The following primary antibodies were used: anti-actin antibody and anti-BTK, anti-pBTK, anti-ITK, anti-GAPDH , anti-IKZF1, and anti-IKZF3 antibodies. All antibodies were used at 1:1000 dilutions in 5% nonfat milk in 1× TBS-T unless otherwise noted in supplier specifications.
Patient primary cells studied in dose–response experiments were treated at densities of 1 × 107 cells plated for 24 h per condition. The baseline and relapsed patient samples were collected from ACD cryovials, thawed, and treated with 1 μM MT-802 24 h before lysis and with 1 μM ibrutinib 2 h before lysis (followed by a 1 h medium washout to simulate in vivo drug metabolism). All primary patient samples were stimulated with anti-IgM 15 min prior to lysis. Primary cell lysates were prepared as previously described. The cell suspension was kept on ice and agitated every 10 min for 30 min, followed by centrifugation for 10 min at 4 °C. Protein quantification was performed for each supernatant using a BCA assay; fifty micrograms of each sample was loaded onto sodium dodecyl sulfate–polyacrylamide gels and electrophoresed. Transfer of the proteins and blocking of membranes were performed as previously described.
Proteins were detected using the following antibodies: anti-phospho-BTK, anti-BTK, and anti-GAPDH. Antibodies used were diluted 1:1000 in Blotto blocker and kept at 4 °C while being constantly agitated for 12–72 h. The blots were washed with 1× TBS-T three times for 10 min while being constantly agitated and then incubated with HRP-conjugated secondary antibodies diluted 1:5000 in 5% nonfat milk in 1× TBS-T for 2 h at 4 °C while being constantly agitated. Prior to their development, the blots were again washed with 1× TBS-T three times for 10 min while being constantly agitated. Blots were developed using one of two chemiluminescent reagents: WesternBright or SuperSignal. Quantification was performed using computer densitometry.

[1]. Targeting the C481S Ibrutinib-Resistance Mutation in Bruton's Tyrosine Kinase Using PROTAC-Mediated Degradation. Biochemistry. 2018 Jul 3;57(26):3564-3575.

Inhibition of Bruton's tyrosine kinase (BTK) with the irreversible inhibitor ibrutinib has emerged as a transformative treatment option for patients with chronic lymphocytic leukemia (CLL) and other B-cell malignancies, yet >80% of CLL patients develop resistance due to a cysteine to serine mutation at the site covalently bound by ibrutinib (C481S). Currently, an effective treatment option for C481S patients exhibiting relapse to ibrutinib does not exist, and these patients have poor outcomes. To address this, we have developed a PROteolysis TArgeting Chimera (PROTAC) that induces degradation of both wild-type and C481S mutant BTK. We selected a lead PROTAC, MT-802, from several candidates on the basis of its potency to induce BTK knockdown. MT-802 recruits BTK to the cereblon E3 ubiquitin ligase complex to trigger BTK ubiquitination and degradation via the proteasome. MT-802 binds fewer off-target kinases than ibrutinib does and retains an equivalent potency (>99% degradation at nanomolar concentrations) against wild-type and C481S BTK. In cells isolated from CLL patients with the C481S mutation, MT-802 is able to reduce the pool of active, phosphorylated BTK whereas ibrutinib cannot. Collectively, these data provide a basis for further preclinical study of BTK PROTACs as a novel strategy for treatment of C481S mutant CLL.[1]

C41H41N9O8

787.8197

787.307

C, 62.51; H, 5.25; N, 16.00; O, 16.25

2231744-29-7

2231744-29-7

138108326

White to off-white solid

2.3

3

13

14

58

1470

0

C, 62.51; H, 5.25; N, 16.00; O, 16.25

AJTLGUJXIKEZCQ-UHFFFAOYSA-N

InChI=1S/C41H41N9O8/c42-37-35-36(25-6-9-29(10-7-25)58-28-4-2-1-3-5-28)47-50(38(35)44-24-43-37)27-14-16-48(17-15-27)18-19-56-20-21-57-23-34(52)45-26-8-11-30-31(22-26)41(55)49(40(30)54)32-12-13-33(51)46-39(32)53/h1-11,22,24,27,32H,12-21,23H2,(H,45,52)(H2,42,43,44)(H,46,51,53)

2-[2-[2-[4-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]ethoxy]ethoxy]-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]acetamide

MT802; MT 802; MT-802; 2231744-29-7; 2-(2-(2-(4-(4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)ethoxy)ethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)acetamide; 2-[2-[2-[4-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]ethoxy]ethoxy]-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]acetamide; 2-[2-(2-{4-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl}ethoxy)ethoxy]-N-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]acetamide; CHEMBL4441907; SCHEMBL21331502; MT-802

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~100 mg/mL (~126.9 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.17 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (3.17 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (3.17 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)