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. 2019 Oct 23;11(3):340–345. doi: 10.1021/acsmedchemlett.9b00399

SGC-AAK1-1: A Chemical Probe Targeting AAK1 and BMP2K

Carrow Wells †,‡, Rafael M Couñago §,∥, Juanita C Limas #, Tuanny L Almeida §,∥, Jeanette Gowen Cook ⊥, David H Drewry †,‡, Jonathan M Elkins §,∇, Opher Gileadi §,∇, Nirav R Kapadia †,‡, Alvaro Lorente-Macias ○, Julie E Pickett †,‡, Alexander Riemen □, Roberta R Ruela-de-Sousa §,∥, Timothy M Willson †,‡, Cunyu Zhang ●, William J Zuercher †,‡,◆, Reena Zutshi □, Alison D Axtman †,‡,*
PMCID: PMC7073879  PMID: 32184967

Abstract

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Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.

Keywords: AAK1, BMP2K, acylaminoindazole, NAK family, endocytosis, protein kinase

Human protein Ser/Thr kinases Adaptor protein 2-Associated Kinase 1 (AAK1) and BMP-2-Inducible Kinase (BMP2K/BIKE) play critical roles in mediating endocytosis and other key signaling pathways. Both are broadly expressed members of the NAK family of human kinases, which also includes Cyclin G-Associated Kinase (GAK) and Myristoylated and Palmitoylated Serine/Threonine Kinase 1 (MPSK1/STK16). The family shares little homology outside of their kinase domains (KDs).1 AAK1 and BMP2K are the most closely related, with overall sequence identity of 50% and KD sequence identity of 74%.2 A key function of AAK1 is regulation of receptor-mediated endocytosis via binding directly to clathrin and phosphorylating the medium subunit of AP2 (adaptor protein 2), which stimulates binding to cargo proteins.3 AAK1 also modulates the Notch pathway, partially through its phosphorylation of Numb.4,5 BMP2K plays a role in osteoblast differentiation, is a clathrin-coated vesicle-associated protein, and, like AAK1, also associates with Numb.6,7

Due to their many functions, AAK1 and BMP2K have been implicated as potential drug targets for diverse conditions. AAK1 has been linked to diseases affecting the brain such as schizophrenia, Parkinson’s disease, and amyotrophic lateral sclerosis as well as implicated as a potential antiviral target for treatment of hepatitis C.8,9 BMP2K has been associated with myopia and evaluated as a potential treatment for HIV.10,11Figure S1 shows the current inhibitor landscape and how their pharmacophores bind to AAK1/BMP2K. Patented AAK1 inhibitors were recently reviewed.12

X-ray crystal structures for the KDs of all NAK family members have been solved and reported.2,13 Published and novel high-resolution crystal structures of AAK1 and BMP2K reveal target-specific structural features that enabled our design of specific chemical probes and allowed further interrogation of the roles that these kinases play.2 Screening a library of kinase inhibitors (Published Kinase Inhibitor Set, PKIS) via differential scanning fluorimetry (DSF) revealed a chemical starting point showing strong AAK1 binding.14 Structure–activity relationships (SAR) were initially established by examining the 3-acylaminoindazole analogs profiled within PKIS (Figures 1 and S2). Next, more than 200 analogs were prepared via the route shown in Scheme S1. A summary of key analogs prepared to arrive at 14 is included in Table 1. Compounds were profiled using the AAK1 split luciferase assay developed by Luceome.15 Potent inhibition of BMP2K was not observed in this assay format.

Figure 1.

Figure 1

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Structures of PKIS hit (1) and key analog 14 with parts of the molecule modified during SAR exploration boxed.

Table 1. SAR of 3-Acylaminoindazole Aryl Ring.

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Cmpd R AAK1 IC50 (nM)22
1 3-NHSO2Me 220
2 4-NHSO2Me 1200
3 3-CONH2 3800
4 4-CONH2 910
5 3-NHAc 1100
6 3-CO2H 18000
7 4-CO2H 2800
8 3-OH 350
9 4-OH 280
10 3-NH2 800
11 3-N(CH3)SO2Me 120
12 3-CH2NHSO2Me 71
13 3-NHSO2(isopropyl) 54
14 3-NHSO2(cyclopropyl) 31
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The effects of aryl substitution on the distal aryl ring were explored, including 3- and 4-position substituents of variable size, electronics, and hydrogen-bonding capacity (Table 1). A sulfonamide was found to be optimal for AAK1 inhibition but only when appended at the 3-position of the aryl ring (1 versus 2). Sulfonamides with small saturated rings (<6 atoms) and short alkyl chains or alkyl amines were the most potent (1, 11, 13, 14). This part of the binding pocket was not tolerant to incorporation of large groups. 3,5-Disubstitution and 3,6-disubstition were disfavored, supporting a small binding pocket for this aryl ring that is sensitive to even minor changes, such as incorporation of a fluorine in place of a hydrogen (data not shown). Finally, activity of 1 and 8 suggests the presence of a hydrogen-bonding network within the ATP-binding pocket that can be accessed by 3-position groups capable of donating a hydrogen. Interestingly, methylation of the aryl 3-nitrogen (11) or insertion of a methylene spacer between the sulfonamide and aryl ring (12) increased potency for AAK1. These results motivated design of a range of sulfonamides to further explore the SAR of this part of the molecule.

The portion of the ATP-binding pocket accessed by the 3-position of the indazole was explored through substitution with a variety of groups (Table 2). Compounds in Table 2 were prepared using the route in Scheme S1, but the step order was reversed. The indazole 3-position substituent appears to form a polar interaction with the kinase hinge region and is sensitive to structural modifications. The alkyl group was varied from cyclopropyl to methyl, isopropyl, cyclobutyl, phenyl, and cyclopropylmethyl to explore the steric bulk tolerated by the pocket. Alternatively, a urea or sulfonamide was incorporated in place of the acylated amine (20, 21, 22) but resulted in loss of AAK1 affinity. The cyclopropanecarboxamide was found to be optimal (14). Minor changes, such as ring opening of the cyclopropane (16) or expansion to a cyclobutane (17), were found to result in loss of AAK1 affinity.

Table 2. SAR of 3-Acylaminoindazole Carboxamide.

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Based on the initial SAR, two additional 3-acylaminoindazoles (23 and 24) were prepared (Table 3) incorporating the aryl 3-cyclopropanecarboxamide that was found to yield potent inhibitors of AAK1. The analogs were designed to probe whether an alkyl amine or longer chain hydrocarbon could be tolerated at the indazole 3-position. Like lead compound 14, these analogs demonstrated promising inhibition of AAK1 in the split luciferase assay (93%I and 94%I of AAK1 at 1 μM for 23 and 24, respectively). TR-FRET binding-displacement assays were employed to further interrogate the biochemical selectivity of these promising 3-aminoacylindazoles across the NAK family (Table 3).21 Compounds 23 and 24 were found to be dual AAK1/BMP2K inhibitors with more than 5-fold and 50-fold selectivity over STK16 and GAK, respectively.

Table 3. NAK Family Binding-Displacement Assay Selectivity Analysis.

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Cmpd R AAK1 Ki (nM)23 BMP2K Ki (nM)23 GAK Ki (nM)23 STK16 Ki (nM)23
14 3-NHSO2(cyclopropyl) 8.3 13 1600 330
23 3-NHSO2N(CH3)2 10 19 2600 350
24 3-NHSO2CH2(cyclopropyl) 6.3 17 870 91
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To understand the selectivity of 23 and 24 across NAK family kinases, cocrystal structures were solved with the BMP2K KD (Figure 2, Table S1). Attempts to obtain cocrystals of acylaminoindazoles bound to the AAK1 KD were not successful. The BMP2K cocrystal structures were solved to ∼2.4 Å resolution and showed that the core indazole formed the critical interaction with the hinge region of the KD.

Figure 2.

Figure 2

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Cocrystal structures of 23 (A) and 24 (B) bound to the BMP2K-KD. Main-chain atoms for residues (131–137) within the BMP2K-KD hinge region, side-chain atoms for BMP2K-KD gatekeeper residue (Met130), and those taking part in polar interactions with the ligands are shown as sticks. Black dashed lines depict possible hydrogen-bonds between protein and ligand atoms. Red spheres denote the position of crystallographic water molecules. The 2Fo–Fc electron density maps (contoured at 1.0σ) are shown below.

Given the high structural and sequence conservation between the ATP-binding sites of the BMP2K and AAK1 KDs (Figures 2 and S3), it is likely that both kinases bind to the indazole inhibitors using common interactions. Thus it is unlikely, using this chemotype, that we can design inhibitors that target only AAK1 or BMP2K. The pendant aryl ring is accommodated by the hydrophobic region adjacent to the gatekeeper residue (residue Met130/126, BMP2K/AAK1 numbering) within the kinase ATP-binding site. These cocrystal structures suggest that large groups attached to the 4-position of the aryl ring (2 and 4) are likely not tolerated due to steric hindrance. Smaller groups at the 3- and 4-positions that can act as hydrogen-bond donors (8 and 9) can make favorable interactions with the DFG motif aspartate residue (Asp198/194), whereas small acidic groups at these positions (6 and 7) are not favored due to charge repulsion. The structures also reveal that a sulfonamide at the 3-position can form hydrogen-bonds with multiple polar residues within or adjacent to the protein ATP-binding pocket (Gln137/133, Asn185/181, and Asp198/194). Groups attached to this sulfonamide are accommodated by a small cavity within the protein P-loop. When considering substituents attached to the indazole ring, poor binding is observed for compounds bearing sulfonamide groups in place of an amide (21 and 22). This finding is due to steric clash and electronic effects caused by the proximity of the sulfonamide oxygen atoms to nearby protein side chains. Compound 20 is proposed to adopt a planar conformation that introduces a steric clash not observed with smaller groups and/or those that are not flat (14 and 17). Cocrystal structures also reveal that groups attached to the 3-position amide are solvent exposed, an observation that helps rationalize the moderate tolerance of AAK1 for larger and bulkier substituents at this position (15, 17, and 19).

Guided by these cocrystal structures, analogs were designed focusing on modifications to the sulfonamide portion of the molecule. Sulfonamides with pendant alkyl chains, alkyl amines, fluorinated alkyl chains, small hydrocarbon-based rings, and fluorinated aryl rings were synthesized. Table 4 includes some of our most promising analogs, the design of which was informed by more than 200 compounds prepared before them, some of which are included herein. Our years of effort on this scaffold enabled us to produce many highly potent compounds. Compounds built on Scaffold B were prepared via Scheme S1. Those built on Scaffold A were prepared using a slightly modified route (Scheme S2). Analogs 25–39 were evaluated for potency and selectivity across the NAK family TR-FRET binding assays. Furthermore, cellular target engagement was determined via NanoBRET (NB) assays, where the respective kinases were fused to 19-kDa luciferase (NLuc) and transiently expressed in HEK293 cells, and then incubated with a cell-permeable fluorescent energy transfer probe (tracer).16 Using increasing concentrations of test compounds, dose-dependent displacement of the tracer was observed, allowing calculation of the IC50 values in Table 4. These NB assays enabled measurement of potency and selectivity of the compounds across the NAK family in living cells (Figure S4).

Table 4. In Vitro and Cellular SAR of 3-Acylaminoindazole Probe Candidates.

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      AAK1 (nM)
 BMP2K(nM)
 GAK (nM)
 STK16 (nM)
  
Cmpd Scaffold R Ki(23) NB IC5024 Ki(23) NB IC5024 Ki(23) NB IC5024a Ki(23) NB IC5024a S10(1 μM)25
14 A cyclopropyl 8.3 641 13 1420 1600 >10000 330 >10000 0.005
25 A N(CH2CH3)2 9.1 770 17 2800 1700 >10000 270 >10000 0.02
26 A N(CH2CH3)(CH3) 8.2 375 20 1890 1700 >10000 310 >10000 0.017
27 A CH2CH(CH3)2 6.2 490 17 1490 1300 >10000 78 >10000 0.002
28 A CH2CH2CH3 12 655 30 2180 1100 9250 110 >10000 0.01
29 A 4-F-benzyl 59 1020 120 2950 7000 9240 430 >10000 0.007
30 A 2-F-benzyl 22 528 51 1620 4600 >10000 470 >10000 0.022
31 A 3-F-benzyl 24 379 53 1190 4600 >10000 510 >10000 0.017
32 A cyclobutyl 8.5 192 17 707 2900 >10000 240 >10000 0.007
33 B CH2CH2CF3 32 2710 27 1890 510 >10000 180 >10000 0.022
34 B N(CH2CF3)(CH3) 44 3400 42 3030 780 >10000 290 >10000  
35 B CH2CF3 39 2350 33 1620 580 >10000 180 >10000 0.005
36 B CH2CH(CH3)2 30 1530 59 6550 3900 >10000 450 >10000 0.002
37 B N(CH2CH3)2 33 1470 74 3830 3200 >10000 220 >10000  
38 B CH2(cyclopropyl) 22 1260 37 2740 3200 >10000 150 >10000 0.01
39 B N(CH2CH3)(CH3) 20 1790 36 2960 360 >10000 86 >10000  
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All analogs (25–39) were found to be dual AAK1/BMP2K inhibitors. Insertion of the methylene spacer between the aryl ring and sulfonamide (Scaffold B) reduced potency for AAK1/BMP2K. Within the Scaffold B group of molecules, incorporation of an alkyl chain capped with fluorines (33, 34, and 35) increased the in vitro biochemical affinity for GAK, but not sufficiently to demonstrate GAK activity in cells (Table S3). In previous work, we had also observed that only low nanomolar inhibitors of GAK possessed cellular activity.17 Nearly all of the analogs were also devoid of activity in the STK16 NB assays. Several single-digit nanomolar biochemical inhibitors of AAK1 were prepared based upon Scaffold A that also demonstrated target engagement in cells with IC50 values between 190–1020 nM. Incorporation of fluorinated aryl rings was tolerated by AAK1 and BMP2K but not by GAK or STK16 (29, 30, and 31).

3-Fluoroaryl analog (31) was one of the most potent compounds in the NB assays for AAK1 and BMP2K, while the 4-fluoro analog (29) was 3-fold less potent on both kinases. Compounds bearing small hydrocarbon chains and rings or alkyl amines on the sulfonamide proved to be the best dual AAK1/BMP2K inhibitors biochemically (14, 25, 26, 27, 28, and 32) and demonstrated at least 35-fold selectivity for BMP2K over GAK. The biochemical selectivity for BMP2K over STK16 was more variable (4–16-fold). When considering the cell-based activity, 14, 26, 27, and 30–32 were the best performers in both AAK1 and BMP2K NB assays, while 25 and 28 exhibited a slight drop in potency when transitioning from the biochemical to cell-based assays. Confirmation of cell penetrance and target engagement enables the facile translation of these compounds to in vivo studies. Furthermore, LipE was calculated for all compounds in Table 4 to estimate their quality as drug candidates (Figure S5).

All compounds from Table 4 built upon Scaffold A and a few representative compounds from Scaffold B (25–33, 35, 36, and 38) were evaluated for selectivity by KINOMEscan (DiscoverX) against 403 wildtype human protein kinases at 1 μM (Table 4).18 The compounds demonstrated remarkable selectivity for AAK1/BMP2K across the 403 human kinases. All proved to be narrow spectrum inhibitors of AAK1/BMP2K with >90% inhibition of only 1–9 kinases (S10(1 μM) = 0.005–0.022, Figure S6).

To explore the phenotypic effect of AAK1/BMP2K inhibition, lead compound 14 and inactive analog 16 were tested in several cell lines. Surprisingly, we observed that 14 had cytostatic effects on cells after 24 h of continuous treatment. Further study in U2OS human osteosarcoma cells showed that 14 elicited cell cycle arrest in G2/M phase at 5–10 μM, which was not captured at the 5 μM dose (Figure S7). Although the molecular mechanism of the cell cycle arrest proved to be elusive, we employed U2OS cells as a screen to remove analogs that displayed this phenotype for consideration as chemical probes. A total of 12 analogs were screened for their effect on G2/M arrest (Figure 3). Nocodazole treatment was used as a positive control for a strong arrest in mitosis. Only analogs 27, 28, and 32 demonstrated the collateral G2 or M phase arrest at 5 μM, while the remaining analogs were devoid of this phenotype (Figures 3 and S8). Potent inhibition of AAK1/BMP2K by arrestors and nonarrestors alike indicated that G2 or M phase arrest was likely due to inhibition of an unidentified off-target kinase.

Figure 3.

Figure 3

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Cell cycle distributions. Cell cycle phases were determined by analytical flow cytometry of DNA content and DNA synthesis in U2OS cells after 24 h treatment with 5 μM of the indicated compounds (n = 3).

Compounds 25 and 26 emerged as probe candidates based on their potency on AAK1 and BMP2K, kinome-wide selectivity, lack of a G2/M arresting phenotype, and LipE values. Following KINOMEscan of compounds 25 (SGC-AAK-1) and 26, the binding affinities (KD) of all kinases inhibited >80% at 1 μM plus AAK1 and a few kinases inhibited by structurally related analogs were determined (Table S4). Only three kinases were bound by 25 within 30-fold of the potency of 25 for AAK1: RIOK1 (KD = 72), RIOK3 (KD = 290), and PIP5K1C (KD = 260).19 Compound 26 bound eight kinases within 30-fold of the potency of AAK1 (Table S4). Most compounds in Table 4 demonstrate potent affinity for RIOK1 and RIOK3 in the corresponding DiscoverX binding assays. Given that RIOK1 and RIOK3 are ATPases, an assay that has ATP present would be much more relevant but has not yet been developed.20 In accordance with our previously described probe criteria,21 SGC-AAK1-1 (25) is a high quality dual AAK1/BMP2K chemical probe. Compound 16 (SGC-AAK1-1N) is the complementary negative control analog. The probe set has been made commercially available, making SGC-AAK1-1 the only AAK1-targeting chemical probe that is widely available alongside its negative control.

We have described the design, synthesis, and biological evaluation of a series of acylaminoindazoles as selective dual inhibitors of AAK1/BMP2K. Compound 25 emerged as our best probe candidate. X-ray cocrystal structures of selected acylaminoindazoles bound to BMP2K revealed key interactions within the ATP binding pocket that explain the high affinity of this series for AAK1 and BMP2K. The effect of 25 on WNT signaling was recently reported.19 Further investigations into effects of selective pharmacological inhibition of AAK1 are ongoing.

Acknowledgments

R.M.C. thanks Diamond Light Source for access to beamline I03 (proposal number MX14664) that contributed to the results presented here. Constructs for NanoBRET measurements of AAK1, BMP2K, GAK, and STK16 were kindly provided by Promega. Dr. Ehrmann from the UNC Mass Spectrometry Core Laboratory provided HRMS support.

Glossary

ABBREVIATIONS

AAK1

AP2-associated protein kinase 1

BMP2K/BIKE

BMP2-inducible kinase

STK16

serine/threonine kinase 16

GAK

cyclin-G-associated kinase

NAK

numb-associated kinase

AP2

adaptor protein 2

TR-FRET

time-resolved fluorescence resonance energy transfer

PKIS

published kinase inhibitor set

DSF

differential scanning fluorimetry

SAR

structure–activity relationships

HEK

human embryonic kidney

NB

NanoBRET

U2OS

U2 osteosarcoma

KD

kinase domain

NLuc

nanoluciferase

tx

treatment

LipE

lipophilic efficiency

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.9b00399.

  • Protein crystallization conditions, NB and cell cycle assay details, experimental methods, and synthesis and characterization of compounds 25, 26, 27, and 28 (PDF)

Accession Codes

PDB codes: compound 23, 5I3O; compound 24, 5I3R.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD 115766], Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP [2013/50724-5, 2014/50897-0, 2016/17469-0], Takeda, and Wellcome [106169/ZZ14/Z]. This work was also supported by the Brazilian agency CNPq [465651/2014-3]. J.G.C. is supported by grants from the NIH/NIGMS (GM083024, and R25GM089569); J.C.L. is supported by an HHMI Gilliam Fellowship for Advanced Study (GT10886) and a T32 award (T32GM007040). The UNC Flow Cytometry Core Facility is supported in part by P30 CA016086 to the UNC LCCC. NC Biotech Center Institutional Support Grants 2017-IDG-1025 and 2018-IDG-1030 and NIH 1UM2AI30836-01 enabled this work. A.L.M. acknowledges support from the Spanish MECD (FPU 14/00818). D.H.D. and R.N. acknowledge 1R44TR001916 for support.

The authors declare no competing financial interest.

Supplementary Material

ml9b00399_si_001.pdf (8.7MB, pdf)

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  22. Each test compound was screened in AAK1 split luciferase assay in duplicate (n = 2) against AAK1 in dose–response (8-pt curve).
  23. Each test compound was screened in duplicate (n = 2) in TR-FRET binding-displacement assay in dose–response (16-pt curve). To allow comparison between different kinases, IC50 values (Table S2) were converted to Ki values using the Cheng–Prusoff equation and the concentration and KD values for the tracer.
  24. AAK1 and BMP2K NB cellular assays were performed in triplicate (n = 3) to provide IC50 values (11-pt curve).
  25. STK16 and GAK NB cellular assays performed in singlicate (n = 1) based on demonstration of poor activity.
  26. Calculation of S10 discussed in Figure S6.

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ml9b00399_si_001.pdf (8.7MB, pdf)

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