doi.org/10.26434/chemrxiv.9756785.v1

A Chemical Probe Targeting AAK1 and BMP2KCarrow Wells, Rafael Counago, Juanita C. Limas, Tuanny L. Almeida, Jeanette G. Cook, David Drewry,Jonathan Elkins, Opher Gileadi, Nirav Kapadia, Álvaro Lorente-Macías, Julie E. Pickett, Alexander J. Riemen,Roberta R. Ruela-de-Sousa, Timothy M. Willson, Cunyu Zhang, William J. Zuercher, Reena Zutshi, Alison D.Axtman

Submitted date: 31/08/2019 • Posted date: 04/09/2019Licence: CC BY-NC-ND 4.0Citation information: Wells, Carrow; Counago, Rafael; Limas, Juanita C.; Almeida, Tuanny L.; Cook, JeanetteG.; Drewry, David; et al. (2019): A Chemical Probe Targeting AAK1 and BMP2K. ChemRxiv. Preprint.

Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2Kinhibitors. Optimization of this 3-acylaminoindazole scaffold 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 inhibitorrepresents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.

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A Chemical Probe Targeting AAK1 and BMP2K Carrow I. Wells,††,¬ Rafael M. Couñago,§,¥ Juanita C. Limas,# Tuanny L. Almeida,§,¥ Jeanette G. Cook,† David H. Drewry,††,¬ Jonathan M. Elkins,§,∧ Opher Gileadi,§,∧ Nirav Kapadia,††,¬ Álvaro Lorente-Ma-cías,• Julie E. Pickett,††,¬ Alexander Riemen,‡ Roberta R. Ruela-de-Sousa,§,¥ Timothy M. Willson,††,¬ Cunyu Zhang,|| William J. Zuercher,††,¬,� Reena Zutshi,‡ Alison D. Axtman*,††,¬ ††Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA ¬Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA §Structural Genomics Consortium, Departamento de Genética e Evolução, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, 13083-886, Brazil ¥Centro de Química Medicinal (CQMED), Centro de Biologia Molecular e Engenharia Genética (CBMEG), UNICAMP, Campinas, SP, 13083-875, Brazil #Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA †Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA ∧Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Re-search Building, Oxford, OX3 7DQ, UK •Departamento de Química Farmacéutica y Orgánica, University of Granada, Granada, 18071, Spain �Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA ‡Luceome Biotechnologies, LLC, Tucson, AZ, 85719, USA ||Platform Technology Sciences, GlaxoSmithKline, Collegeville, PA, 19426, USA

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

ABSTRACT: Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization of this 3-acylaminoindazole scaffold 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 func-tionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.

The human protein Ser/Thr kinases Adaptor protein 2-Associ-ated 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 and are 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.1 AAK1 and BMP2K are the most closely related, with overall sequence identity of 50% and kinase do-main sequence identity of 74%.2 A key function of AAK1 is regulation of receptor-mediated endocytosis via binding di-rectly to clathrin and phosphorylating the medium subunit of AP2 (adaptor protein 2), which stimulates binding to cargo pro-teins.3-5 AAK1 also modulates the Notch pathway, partially through its phosphorylation of Numb.6, 7 BMP2K plays a role in

osteoblast differentiation, is a clathrin-coated vesicle-associated protein, and, like AAK1, also associates with Numb.8, 9

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 anti-viral target for the treatment of Hepatitis C.5, 10, 11 BMP2K has been associated with myopia and evaluated as a potential treatment for HIV.12,

13 A dual AAK1/BMP2K small molecule inhibitor was recently reported as a novel therapeutic to treat neuropathic pain.14

X-ray crystal structures for the kinase domains of all NAK family members have been solved and reported.2, 15, 16 Published and novel high-resolution crystal structures of AAK1 and BMP2K reveal target-specific structural features that have ena-bled our design of specific chemical probes and allowed further

interrogation of the roles that these kinases play.2 Through screening a library of kinase inhibitors (Published Kinase Inhib-itor Set, PKIS) via differential scanning fluorimetry (DSF), a chemical starting point showing strong AAK1 binding was identified (Figure 1).17 Structure–activity relationships (SAR) were initially established by examining the 3-acylami-noindazole analogs profiled within PKIS. Next, more than 200 analogs were prepared via the route shown in Scheme 1. Briefly, acylation of the indazole aniline with cyclopropanecar-bonyl chloride furnished the key intermediate, which was then subjected to Suzuki coupling with a boronic acid. Finally, free anilines on intermediates were reacted with sulfonyl chlorides to yield the desired sulfonamide products. A summary of key analogs prepared to arrive at 14 is included in Table 1. The com-pounds were profiled using the AAK1 split luciferase assay de-veloped by Luceome.18, 19 1–14 also showed similar affinity for BMP2K (data not shown).

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

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. Sulfona-mides, amines, amides, anilines, phenols, acids, and other groups were incorporated in these positions (Table 1). A sulfon-amide was found to be optimal in terms of AAK1 inhibition but only when appended at the 3-position of the aryl ring (1 versus 2). Sulfonamides with small saturated rings of 5 atoms or fewer 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. In addition, 3,5-disubstitution or 3,6-disubstition was disfavored, supporting a small pocket for binding of 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, the 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) did not decrease potency for AAK1 but rather in-creased it. These results motivated the design of a range of sul-fonamides to further explore the SAR of this part of the mole-cule. Scheme 1. Route to prepare compounds in Table 1.a

aReagents and conditions: (a) Cyclopropanecarbonyl chloride, pyr-idine, 0°C (68-83%) (b) R-boronic acid pinacol ester, Pd(dppf)Cl2, Na2CO3, dioxane, 120°C (82-100%) (c) R’-sulfonyl chloride, pyri-dine, 0°C (7-100%).

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 pre-pared using the route shown in Scheme 1, but the order of the steps was reversed. Thus, a Suzuki reaction was first carried out, and then the indazole aniline was reacted with the correspond-ing acylating or sulfonylating groups to generate the analogs in Table 2. 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 cy-clopropyl to methyl, isopropyl, cyclobutyl, phenyl, and cyclo-propylmethyl 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 opti-mal (14). Minor changes, such as ring opening of the cyclopro-pane (16) or expansion to a cyclobutane (17) were found to re-sult in modest loss of AAK1 affinity. Table 1. SAR of 3-acylaminoindazole aryl ring.

Cmpd R AAK1 IC50 (nM)1

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

1Each test compound was screened in AAK1 split luciferase as-say in duplicate (n=2) against AAK1 in dose-response (8-pt curve).

Based on the initial SAR, two additional 3-acylami-noindazoles (23 and 24) were prepared (Table 3) incorporating the aryl 3-cyclopropanecarboxamide that was found to yield po-tent 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 and BMP2K in the split luciferase assay (93%I and 94%I of AAK1 at 1 μM for 23 and 24, respectively). TR-FRET binding-dis-placement assays were employed to further interrogate the bio-chemical selectivity of these promising 3-aminoacylindazoles across the four NAK family members (Table 3).21 23 and 24 were found to be dual AAK1/BMP2K inhibitors with more than 5-fold selectivity over STK16 and more than 50-fold selectivity over GAK.

To understand the selectivity of 23 and 24 across the NAK family kinases, co-crystal structures were solved with the BMP2K kinase domain (KD, Figure 2, Table S1). The BMP2K co-crystal structures were solved to ~2.4 Å resolution and

NH

N

HN

HN

O

SO

O

1AAK1 split luciferase IC50 = 220 nM

NNH

HN

HN

SO

O

O

14AAK1 split luciferase IC50 = 31 nM

TR-FRET AAK1 Ki = 8.3 nMTR-FRET BMP2K Ki = 13 nM

NH

NBr

NH2a

NH

NBr

NH

O

b

NH

N

HNO

R

I IIR = NH2 or CH2NH2R = NHSO2R’ or CH2NHSO2R’c

NNH

NH

R

O

showed that the core indazole formed the critical interaction with the hinge region of the kinase domain.

Table 2. SAR of 3-acylaminoindazole carboxamide.

Cmpd R AAK1 IC50 (nM)1

14

31

15

3500

16

18000

17

370

18

560

19

2200

20

>50000

21

>50000

22

7700

1Each test compound was screened in duplicate (n=2) in AAK1 split luciferase assay in dose-response (8-pt curve).

Given the high structural and sequence conservation be-tween the ATP-binding sites of the BMP2K and AAK1 kinase domains (Figure 2), it is likely that both kinases bind to the in-dazole inhibitors using common interactions. 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 co-crys-tal structures suggest that large groups attached to the 4-position of the aryl ring (such as in 2 and 4) are likely not tolerated due to steric hinderance. Smaller groups at the 3- and 4-positions that can act as H-bond donors (such as in 8 and 9) can make favorable interactions with the DFG motif aspartate residue (Asp198/194, BMP2K/AAK1 numbering), whereas small acidic groups at these positions (such as in 6 and 7) are not fa-vored due to charge repulsion. The structures also reveal that a sulfonamide at the 3-position can form H-bonds with multiple polar residues within or adjacent to the protein ATP-binding pocket (Gln137/133, Asn185/181 and Asp198/194, BMP2K/AAK1 numbering). Groups attached to this sulfona-mide 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 sulfona-mide groups in place of an amide (21 and 22). This finding is due to steric clash and electronic effects caused by the proxim-ity of the sulfonamide oxygen atoms to nearby protein side chains. The co-crystal structures also reveal that groups at-tached to the 3-position amide are solvent exposed, an observa-tion that helps rationalize the moderate tolerance of AAK1 for larger and bulkier substituents at this position (15, 17 and 19).

Guided by the co-crystal structures, analogs were designed with a focus 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). Compounds built on Scaffold B were prepared using the chem-istry in Scheme 1. Those built on Scaffold A were prepared us-ing a slightly modified route. Briefly, reaction of the 3-amino-phenylboronic acid pinacol ester with respective sulfonyl chlo-rides to furnish the sulfonamide-bearing boronic acid pinacol esters was found to be high yielding. This intermediate was cou-pled to the acylated indazole (I), prepared in Scheme 1, using Suzuki conditions. The analogs (25–39) were evaluated for po-tency and selectivity across the NAK family TR-FRET binding assays. Furthermore, cellular target engagement of AAK1, BMP2K, GAK and STK16 were determined by 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).20 Using increasing concentrations of test compounds dose-dependent displacement of tracer was ob-served, 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 S1).

Table 3. NAK family TR-FRET data.

1Each test compound was screened in duplicate (n=2) in dose-response (16-pt curve). IC50 values were converted to Ki values using the Cheng Prusoff equation and the concentration and KD values for the tracer.

NNH

R

HN

SO

O

NH

O

NH

O

NH

O

NH

O

NH

O

NH

O

NH

O

N

NH

SO O

NH

SO O

Cmpd R AAK1 Ki

(nM)1 BMP2K Ki

(nM)1 GAK Ki

(nM)1 STK16 Ki

(nM)1 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

NNH

NH

R

O

Figure 2. Co-crystal 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 are represented as sticks. The side-chain atoms for BMP2K-KD gatekeeper reside (Met130) and those tak-ing 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 crystallo-graphic water molecules. The bottom portion of each panel shows the chemical structure and 2Fo-Fc electron density maps (con-toured at 1.0s) for each ligand. Active site sequence alignment for

NAK family included below with non-identical AAK1/BMP2K residues bolded and underlined. Residue numbers shown on the top row are for BMP2K.

Analysis of the data from both the biochemical and cellular assays revealed some notable trends and highlighted promising compounds as potential chemical probes. All of the analogs (25–39) were found to be dual AAK1/BMP2K inhibitors. Inser-tion of the methylene spacer between the aryl ring and sulfona-mide (scaffold B) reduced potency for AAK1/BMP2K. Within the scaffold B group of molecules, incorporation of an alkyl chain capped with fluorines (33, 34, 35) increased the in vitro biochemical affinity for GAK, but not sufficiently to demon-strate GAK activity in cells (Table S2). In previous work, we had also observed that only low nanomolar inhibitors of GAK possessed cellular activity.21 Almost 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 engage-ment 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, 31). Scheme 2. Route to prepare compounds in Tables 3 and 4.a

aReagents and conditions: (a) R-sulfonylchloride, pyridine, 0°C (49-65%) (b) I, Pd(dppf)Cl2, Na2CO3, dioxane, 120°C (36-66%).

Table 4. SAR of 3-acylaminoindazole probe candidates.

1Each analog was screened in the TR-FRET in duplicate (n=2) and dose-response (16-pt curve). IC50 values were converted to Ki values using the Cheng Prusoff equation and the concentration and KD values for the tracer. 2AAK1 and BMP2K NB assays were performed in triplicate (n=3) to provide IC50 values (11-pt curve). 3STK16 and GAK NB assays performed in singlicate (n=1) based on demonstration of poor activity. 4S10(1 μM) is a measure of selectivity equal to the percentage of the screenable human kinome biochemically inhibited by >90% at 1 μM.

aBH2NO

O BO

OHN

SO

OR

b NNH

HN

HN

O

SO

OR

Cmpd Scaffold R AAK1 (nM) BMP2K (nM) GAK (nM) STK16 (nM)

S10(1 μM) Ki1 IC502 Ki1 IC502 Ki1 IC503 Ki1 IC503

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 NT 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 NT 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 NT

5

The 3-fluoroaryl analog (31) was one of the most potent compounds in the cellular NB assays for AAK1 and BMP2K, while the 4-fluoro analog (29) was much less active 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, 32) and demonstrated at least 35-fold selectivity for BMP2K over GAK. The biochemical selectivity for BMP2K over STK16 was more variable, ranging from 4–16-fold. When considering the cell-based activity, 14, 26, 27, and 30–32 were the best performers in both the AAK1 and BMP2K NB, while 25 and 28 exhibited a slight drop in potency when transitioning from the biochemical to cell-based assays.

Figure 3. Cell cycle distributions. Cell cycle phases were deter-mined by analytical flow cytometry of DNA content and DNA syn-thesis in U2OS cells after 24h treatment with 5 µM of the indicated compounds (error bars represent n=3). Note: compounds 27, 28, and 32 induce G2/M arrest (nocodazole serves as a positive control for arrest with G2/M DNA content).

All of the 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).22 The compounds demonstrated remarkable se-lectivity for AAK1/BMP2K across the 403 human kinases. 27 and 36 were found to selectivity inhibit only AAK1/BMP2K (S10(1 µM) = 0.002). The remaining analogs were all narrow spectrum inhibitors of AAK1/BMP2K with >90% inhibition of only 1–8 additional kinases (S10(1 µM) = 0.005–0.01, Figure S4).

To explore the phenotypic effect of AAK1/BMP2K inhibi-tion, the lead compound 14 and the inactive analog 16 were tested in several cell lines. Surprisingly, we observed that 14 had cytostatic effects on cells after 24h 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 (Figure S2). The lack of effect of inactive analog 16 implied that the G2/M arrest was not due to inhibition of AAK1/BMP2K but was likely due to inhibition of an off-target kinase. Cell growth inhibition and cell cycle arrest were observed in HEK293T cells as well (data not shown). Although the molecular mechanism of the cell cycle arrest proved to be elusive, we employed the 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). Nocondazole 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 (Figure 3, Figure S3). Compound 25 was selected as a chemical probe due to its potency on AAK1 and BMP2K, its kinome-wide selectivity, and its lack of a G2/M arresting phenotype.

Following KINOMEscan of compound 25 (SGC-AAK-1), the binding affinity (KD) of all kinases inhibited >80% at 1 μM plus AAK1 and a few kinases inhibited by structurally related analogs was determined (Table S3). Only 3 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).23 In accordance with our previously described probe criteria,24 SGC-AAK1-1 (25) is the best available dual AAK1/BMP2K chemical probe. Compound 16 (SGC-AAK1-1N) is the compli-mentary negative control analog.

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 the most selective and cell active compound within the series. X-ray co-crystal 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 re-ported.23 Further investigations into the effects of selective pharmacological inhibition of AAK1 are ongoing.

ASSOCIATED CONTENT Supporting Information Supplemental material is available free of charge via the Internet at http://pubs.acs.org. Protein crystallization conditions, NanoBRET and cell cycle assay results, experimental methods, synthesis and characterization of target compounds 25, 26, 27, and 28 are included (PDF).

Accession Codes The PDB accession codes for the X-ray co-crystal structures of BMP2K + 23 and BMP2K + 24 are 5I3O and 5I3R, respectively.

AUTHOR INFORMATION Corresponding Author * Tel: 919-962-5349. E-mail: alison.axtman@unc.edu.

ORCID Carrow I. Wells: 0000-0003-4799-6792 Rafael M. Couñago: 0000-0002-2826-1689 Juanita C. Limas: 0000-0001-6384-8787 Tuanny L. Almeida: 0000-0002-4528-9130 Jeanette G. Cook: 0000-0003-0849-7405 David H. Drewry: 0000-0001-5973-5798 Jonathan M. Elkins: 0000-0002-2826-1689 Opher Gileadi: 0000-0002-2826-1689 Nirav Kapadia: 0000-0002-2826-1689 Álvaro Lorente-Macías: 0000-0001-9510-714X Julie E. Pickett: 0000-0002-9535-8528

6

Roberta R. Ruela-de-Sousa: 0000-0002-3762-2247 Timothy M. Willson: 0000-0003-4181-8223 William J. Zuercher: 0000-0002-9836-0068 Alison D. Axtman: 0000-0003-4779-9932

Notes The authors declare no competing financial interest.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manu-script.

Funding Sources The Structural Genomics Consortium is a registered charity (num-ber 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 grant no. 115766], Janssen, Merck KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario Ministry of Economic Development and In-novation, Pfizer, São Paulo Research Foundation-FAPESP [2013/50724-5, 2014/5087-0 and 2016/17469-0], Takeda, and Wellcome [106169/ZZ14/Z]. This work was also supported by the Brazilian agency CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) [465651/2014-3]. J.G.C is supported by grants from the NIH/ NIGMS (GM083024, and R25GM089569); JCL is supported by an HHMI Gilliam Fellowship for Advanced Study (GT10886) & T32 award (T32GM007040). The UNC Flow Cytometry Core Facility is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Com-prehensive Cancer Center. Research reported in this publication was supported in part by the NC Biotech Center Institutional Sup-port Grants 2017-IDG-1025 and 2018-IDG-1030, and by the NIH 1UM2AI30836-01. A.L.M. acknowledges support from the Span-ish Ministry of Education, Culture and Sports (FPU grant ref. 14/00818). D.H.D. and R.N. acknowledge 1R44TR001916 for sup-port.

ACKNOWLEDGEMENT R.M.C. thanks Diamond Light Source for access to beamline I03 (proposal number MX14664) that contributed to the results pre-sented here. Constructs for NanoBRET measurements of AAK1, BMP2K, GAK and STK16 were kindly provided by Promega.

ABBREVIATIONS AAK1, AP2-associated protein kinase 1; BMP2K/BIKE, BMP2-inducible kinase; STK16, serine/Threonine kinase 16; GAK, cy-clin-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, differ-ential scanning fluorimetry; ATP, adenosine 5’-triphosphate; SAR, structure–activity relationship; HEK, human embryonic kidney; NB, NanoBRET; U2OS, U2 osteosarcoma; KD, kinase domain; NLuc, Nanoluciferase; tx, treatment; H-bond, hydrogen-bond.

REFERENCE 1. Smythe, E.; Ayscough, K. R., The Ark1/Prk1 family of protein kinases. Regulators of endocytosis and the actin skeleton. EMBO reports 2003, 4 (3), 246-51. 2. Sorrell, F. J.; Szklarz, M.; Abdul Azeez, K. R.; Elkins, J. M.; Knapp, S., Family-wide Structural Analysis of Human Numb-Associated Protein Kinases. Structure 2016, 24 (3), 401-11. 3. Conner, S. D.; Schmid, S. L., Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-

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7

MPSK1 reveals an atypical activation loop architecture. Structure 2008, 16 (1), 115-24. 17. Elkins, J. M.; Fedele, V.; Szklarz, M.; Abdul Azeez, K. R.; Salah, E.; Mikolajczyk, J.; Romanov, S.; Sepetov, N.; Huang, X. P.; Roth, B. L.; Al Haj Zen, A.; Fourches, D.; Muratov, E.; Tropsha, A.; Morris, J.; Teicher, B. A.; Kunkel, M.; Polley, E.; Lackey, K. E.; Atkinson, F. L.; Overington, J. P.; Bamborough, P.; Muller, S.; Price, D. J.; Willson, T. M.; Drewry, D. H.; Knapp, S.; Zuercher, W. J., Comprehensive characterization of the Published Kinase Inhibitor Set. Nature biotechnology 2016, 34 (1), 95-103. 18. Jester, B. W.; Cox, K. J.; Gaj, A.; Shomin, C. D.; Porter, J. R.; Ghosh, I., A coiled-coil enabled split-luciferase three-hybrid system: applied toward profiling inhibitors of protein kinases. J Am Chem Soc 2010, 132 (33), 11727-35. 19. Jester, B. W.; Gaj, A.; Shomin, C. D.; Cox, K. J.; Ghosh, I., Testing the promiscuity of commercial kinase inhibitors against the AGC kinase group using a split-luciferase screen. Journal of medicinal chemistry 2012, 55 (4), 1526-37. 20. Vasta, J. D.; Corona, C. R.; Wilkinson, J.; Zimprich, C. A.; Hartnett, J. R.; Ingold, M. R.; Zimmerman, K.; Machleidt, T.; Kirkland, T. A.; Huwiler, K. G.; Ohana, R. F.; Slater, M.; Otto, P.; Cong, M.; Wells, C. I.; Berger, B. T.; Hanke, T.; Glas, C.; Ding, K.; Drewry, D. H.; Huber, K. V. M.; Willson, T. M.; Knapp, S.; Muller, S.; Meisenheimer, P. L.; Fan, F.; Wood, K. V.; Robers, M. B., Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement. Cell chemical biology 2017, 25 (2), 206-214.

21. Asquith, C. R. M.; Laitinen, T.; Bennett, J. M.; Godoi, P. H.; East, M. P.; Tizzard, G. J.; Graves, L. M.; Johnson, G. L.; Dornsife, R. E.; Wells, C. I.; Elkins, J. M.; Willson, T. M.; Zuercher, W. J., Identification and Optimization of 4-Anilinoquinolines as Inhibitors of Cyclin G Associated Kinase. ChemMedChem 2018, 13 (1), 48-66. 22. Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L. M.; Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P., Comprehensive analysis of kinase inhibitor selectivity. Nature biotechnology 2011, 29 (11), 1046-51. 23. Agajanian, M. J.; Walker, M. P.; Axtman, A. D.; Ruela-de-Sousa, R. R.; Serafin, D. S.; Rabinowitz, A. D.; Graham, D. M.; Ryan, M. B.; Tamir, T.; Nakamichi, Y.; Gammons, M. V.; Bennett, J. M.; Counago, R. M.; Drewry, D. H.; Elkins, J. M.; Gileadi, C.; Gildadi, O.; Godoi, P. H.; Kapadia, N.; Muller, S.; Santiago, A. S.; Sorrell, F. J.; Wells, C. I.; Fedorov, O.; Willson, T. M.; Zuercher, W. J.; Major, M. B., WNT activates the AAK1 kinase to promote clathrin-mediated endocytosis of LRP6 and establish a negative feedback loop. Cell reports 2019, 26 (1), 79-83. 24. Asquith, C. R. M.; Berger, B.-T.; Wan, J.; Bennett, J. M.; Capuzzi, S. J.; Crona, D. J.; Drewry, D. H.; East, M. P.; Elkins, J. M.; Fedorov, O.; Godoi, P. H.; Hunter, D. M.; Knapp, S.; Müller, S.; Torrice, C. D.; Wells, C. I.; Earp, H. S.; Willson, T. M.; Zuercher, W. J., SGC-GAK-1: A Chemical Probe for Cyclin G Associated Kinase (GAK). Journal of medicinal chemistry 2019, 62 (5), 2830-2836.

download fileview on ChemRxivAcylaminoindazole MCL formatted final.pdf (1.01 MiB)

SUPPLEMENTARY FIGURES AND TABLES CRYSTALLOGRAPHY, and BIOLOGICAL SCREENING Supplementary Table 1. Crystal structure data collection and refinement statistics.

Ligand 5I3O 5I3R Data collection X-ray source DLS I03 DLS I03 Wavelength (Å) 0.97626 0.97626 Space group P43212 P43212 Cell dimensions a=b, c (Å) α=β=γ (o)

78.3, 255.4 90.0

78.3, 255.4 90.0

Resolution (Å)* 29.56-2.40 (2.50-2.40)

29.56-2.40 (2.49-2.40)

No. of unique reflections*

31,839 (3,167) 32,194 (3,282)

Rmerge (%)* 23.4 (478.3) 12.2 (129.5) Mean I/σI * Mean CC(1/2)*

7.0 (0.3) 0.99 (0.1)

9.1 (1.3) 0.99 (0.6)

Completeness (%)* 99.5 (96.6) 99.8 (99.7) Redundancy* 6.3 (5.0) 5.0 (5.2) Refinement Resolution (Å) 24.71-2.40 (2.48-

2.40) 21.71-2.40 (2.48-

2.40) Rcryst / Rfree (%) 18.2 / 22.1 18.8 / 23.1 No. of atoms / Mean B-factor (Å)

Protein atoms 4,757 (67.7) 4,735 (59.8) Solvent atoms 293 (62.5) 202 (57.7) Ligand atoms 56 (63.2) 58 (55.0) Rmsd bond lengths (Å) 0.010 0.010 Rmsd bong angles (o) 1.13 1.09 Ramachandran statistics (%)

Favored 97.0 97.2 Allowed 3.0 2.6 Outlier 0.0 0.2 PDB ID 5I3O 5I3R

Crystallization conditions

20% PEG 8000, 0.2M ammonium

chloride, 10% ethylene glycol, 0.1M MES pH 6

20% PEG 8000, 0.2 M ammonium chloride, 10%

ethylene glycol, 0.1M MES pH 6.0

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Compound 14

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Compound 25

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AAK1

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Compound 27

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GAK

STK16

AAK1

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Compound 28

STK16GAKBMP2KAAK1

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Compound 29

BMP2K

GAK

STK16

AAK1

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Compound 31

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STK16

GAK

AAK1

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Compound 26

STK16

BMP2K

AAK1

GAK

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Compound 30

GAKSTK16

BMP2K

AAK1

Supplementary Figure 1. NAK family nanoBRET assay curves for all compounds in Table 4.

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Compound 32

STK16

GAK

BMP2K

AAK1

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Compound 33

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STK16

GAK

BMP2KAAK1

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Compound 34

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BMP2K

GAKSTK16

AAK1

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Compound 35

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STK16GAKBMP2KAAK1

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Compound 36

GAK

STK16

BMP2K

AAK1

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Compound 37

BMP2K

STK16GAK

AAK1

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Compound 38

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GAK

STK16

BMP2KAAK1

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Compound 39

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BMP2K

STK16

GAK

AAK1

Supplementary Table 2. Additional GAK nanoBRET data.1

1GAK NB assays performed in singlicate (n=1).

Supplementary Figure 2. Dose-response DAPI/EdU plots for compound 14. Asynchronously proliferating U2OS cells were pulse-labeled with EdU to measure DNA synthesis and with DAPI for DNA content.

EdU

DNA content

1µM 10µM

DNA content

EdU

5µM

DNA content

EdU

Compound GAK NB IC50 (nM)

%Inh of GAK at 10 µM

14 >10,000 - 23 >10,000 - 25 >10,000 - 26 >10,000 10% 27 >10,000 - 28 9253 23% 29 9235 - 30 >10,000 - 31 >10,000 17% 32 >10,000 15% 33 >10,000 51% 34 >10,000 - 35 >10,000 55% 36 >10,000 - 37 >10,000 19% 38 >10,000 26% 39 >10,000 20%

Supplementary Figure 3. Representative DAPI/EdU plots for asynchronously proliferating U2OS cells after 24 h treatment with each compound at 5 μM or with 33 mM nocodazole (Sigma). Cells were pulse-labeled with EdU to measure DNA synthesis and with DAPI for DNA content. The proportion of cells in each cell cycle phase is indicated.

Supplementary Figure 4. Treespots of compounds from Table 4 showing kinases inhibited at or below 10% of control at DiscoverX.

Supplementary Table 3. Select inhibition and KD follow-up data collected at DiscoverX for compound 25 (SGC-AAK1-1).

Kinase %I at 1 μM KD (nM)1 PIP5K1C 100 260

RIPK2 100 10000 TNNI3K 100 10000

CLK4 100 10000 PRKCH 100 10000

BIKE/BMP2K 95.6 930 RPS6KA5(Kin.Dom.1-N-terminal) 95.5 10000

FGFR4 90.1 10000 RIOK1 86 72 AAK1 76 26 SRPK3 61 3100 RIOK3 56 290 CDKL2 52 880 MYLK2 28 960

1An 11-pt curve (n=1) was generated and KD was calculated using the Hill equation. Cloning, expression, purification and crystallization. BMP2K38-345 (K320A, K321A) with a tobacco etch virus (TEV) protease cleavable, N-terminal 6xHis tag was expressed from vector pNIC-ZB. To improve BMP2K crystallization, a cluster of surface entropy reduction mutations 1 was engineered into the expression construct, K320A/K321A.2 For protein production, the host E. coli strain, BL21(DE3)-R3 expressing lambda phosphatase, was cultivated in TB medium (+ 50 µg/ml kanamycin, 35 µg/ml chloramphenicol) at 37 oC until OD600 reached ~3 and then cooled to 18 oC for 1 h. Isopropyl 1-thio-D-galactopyranoside was added to 0.1 mM, and growth continued at 18 oC overnight. The cells were collected by centrifugation then resuspended in 2x lysis buffer (100 mM HEPES buffer, pH 7.5, 1.0 M NaCl, 20 mM imidazole, 1.0 mM tris(2-carboxyethyl)phosphine, 2x Protease Inhibitors Cocktail Set VII (Calbiochem, 1/1000 dilution) and flash-frozen in liquid nitrogen. Cells were lysed by sonication on ice. The resulting proteins were purified using Ni-Sepharose resin (GE Healthcare) and eluted stepwise in binding buffer with 300 mM imidazole. Removal of the hexahistidine tag was performed at 4 oC overnight using recombinant TEV protease. Proteins were further purified using reverse affinity chromatography on Ni-Sepharose followed by gel filtration (Superdex 200 16/60, GE Healthcare). Protein in gel filtration buffer (25 mM HEPES, 500 mM NaCl, 0.5 mM TCEP, 5% [v/v] glycerol) was concentrated to 12 mg/ml using 30 kDa MWCO centrifugal concentrators (Millipore) at 4 oC. 3-Acylaminoindazole 1 in 100 % DMSO was added to the protein in a 3-fold molar excess and incubated on ice for approximately 30 min. The mixture was centrifuged at 14,000 rpm for 10 min at 4 oC prior to setting up 150 nL volume sitting drops at three ratios (2:1, 1:1, or 1:2 protein-inhibitor complex to reservoir solution). Crystallization experiments were performed at 20 °C. Crystals were cryoprotected in mother liquor supplemented with 20-25 % glycerol before flash-freezing in liquid nitrogen for data collection. Diffraction data were collected at the Diamond Light Source. The best diffracting crystals grew under the conditions described in Table 1. When noted, crystal optimization used Newman’s buffer system 3 and defined PEG smears.4

Structure Solution and Refinement. Diffraction data were integrated using XDS 5 and scaled using AIMLESS from the CCP4 software suite.6 Molecular replacement for was performed with Phaser 7 using BMP2K/AZD7762 (PDB: 4W9W) 2 as a search model. Automated model building was performed with Buccanner.8 Automated refinement was performed in PHENIX.9 Coot 10 was used for manual model building and refinement. Structure validation was performed using MolProbity.11 Structure factors and coordinates have been deposited in the PDB (see Table 1). Computation. Solvation free energies and quantum mechanical calculations were run using Macromodel (12) and Jaguar (13) from Schrodinger Inc. Further computational studies were performed using Maestro 11 from Schrodinger (Schrödinger Release 2016-4: Maestro, Schrödinger, LLC, New York, NY, 2016). The lowest energy configurations for 23 and 24 at pH = 7 were generated with the Ligand Preparation application (Schrödinger Release 2016-4: LigPrep, Schrödinger, LLC, New York, NY, 2016). Next, PDB crystal structures for AAK1, BMP2K and the other kinases in Table 4 were downloaded and prepared for docking. Briefly, bond orders were assigned, hydrogen atoms were added, selenomethionine residues were converted to methionines, and missing side chains were filled with the Prime application. The protein grid for docking was set by selecting the center of the cocrystal ligand in the PDB structure. The Glide application was used for docking of 1 and 2 to all kinases (Schrödinger Release 2016-4: Glide, Schrödinger, LLC, New York, NY, 2016.). Flexible ligand sampling was allowed with the XP (extra precision) scoring function (http://pubs.acs.org/doi/abs/10.1021/jm051256o). Once the ligands were docked and the lowest energy poses were retrieved, a Rigid Rotor Harmonic Oscillation (RRHO) approximation was performed on the theoretical protein-ligand complex (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3329805/). For the RRHO approximation, an OPLS_2005 force field was applied (http://pubs.acs.org/doi/abs/10.1021/acs.jctc.5b00864). Split luciferase AAK1 assay: KinaseSeeker is a homogeneous competition binding assay where the displacement of an active site dependent probe by an inhibitor is measured by a change in luminescence signal. Luminescence readout translates into a highly sensitive and robust assay with low background and minimal interference from test compounds.14-15 10 mM stock solutions of the compounds were serially diluted in DMSO to make assay stocks. Prior to initiating IC50 determinations, the test compounds were evaluated for false positive against split-luciferase. Each test compound was screened in duplicate against AAK1 at 8 different concentrations. For kinase assays, Cfluc-kinase was translated along with Fos-Nfluc using a cell-free system (rabbit reticulocyte lysate) at 30oC for 90 min. 24 μL aliquot of this lysate containing either 1 μL of DMSO (for no-inhibitor control) or compound solution in DMSO was incubated for 30 minutes at room temperature followed by 1 hour in presence of a kinase specific probe. 80 μL of luciferin assay reagent was added to each solution and luminescence was immediately measured on a luminometer. The % Inhibition and % Activity Remaining was calculated using the following equation:

% Inhibition = ALUControl – ALUSample x 100

ALUControl

% Activity Remaining = 100 - % Inhibition

The % Activity was plotted against compound concentration and the IC50 was determined for each compound using an 8-point curve. The IC50 of a known inhibitor, Sunitinib, against AAK1 in our assay was used as a reference. Binding-displacement assays: The TR-FRET ligand binding-displacement assays for AAK1, BMP2K, GAK and STK16 were performed as previously described.16 Kinome screening: The KINOMEscan assay panel was measured at DiscoverX Corporation as previously described.17 Cell culture: U2OS cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2 mM L-glutamine, 1x pen-strep, and 10% fetal bovine serum (FBS). Cells were incubated in 5% CO2 at 37˚C. Cells lines were passaged every 72 hours with trypsin and not allowed to reach confluency. HEK293 cells were cultured as described previously.18 NanoBRET measurements: Constructs for NanoBRET measurements of AAK1, BMP2K, GAK and STK16 were kindly provided by Promega. Example protocol for AAK1: The N-terminal Nano Luciferase/AAK1 fusion (NL-AAK1) was encoded in pFN31K expression vector, including flexible Gly-Ser-Ser-Gly linkers between NanoLuc and AAK1(Promega). For cellular NanoBRET target engagement experiments, a 10 µg/mL solution of DNA in Opti-MEM without serum was made containing 9 μg/mL of Carrier DNA (Promega) and 1 μg/mL of NL-AAK1 for a total volume of 1.05 mL. To this solution was then added 31.5 µL of FuGENE HD (Promega) to form a lipid:DNA complex. The solution was then mixed by inversion 8 times and incubated at room temperature for 20 min. The resulting transfection complex (1.082 mL) was then gently mixed with 21 mL of HEK-293 cells (ATCC) suspended at a density of 2 x 105 cells/mL in DMEM (Gibco) + 10% FBS (Corning). This solution was then dispensed (100 μL) into 96-well tissue culture treated plates (Corning #3917) followed by incubation (37 °C / 5 % CO2) for 24 h. After 24 hours the media was removed and replaced with 85 μL of room temperature Opti-MEM without phenol red. NanoBRET Tracer K5 (Promega) was used at a final concentration of 1.0 μM as previously determined to be the optimal concentration in a titration experiment. A total of 5 μL per well (20x working stock of NanoBRET Tracer K5 [20 μM] in Tracer Dilution Buffer (Promega N291B) was added to all wells, except the “no tracer” control wells. All test compounds were prepared initially as concentrated (10 mM) stock solutions in 100% DMSO (Sigma), then diluted in Opti-MEM media (99%) to prepare 1 % DMSO working stock solutions. A total of 10 μL per well of the 10-fold test compound stock solutions (final assay concentration 0.1 % DMSO) were added. For “no compound” and “no tracer” control wells, a total of 10 µL per well of Opti-MEM plus DMSO (9 µL Opti-MEM with 1 µL DMSO) was added, final concentration 1 % DMSO). 96-well plates containing cells with NanoBRET Tracer K5 and test compounds (100 µL total volume per well) were equilibrated (37 °C / 5 % CO2) for 2 h.

After 2 hours the plates were cooled to room temperature for 15 min. To measure NanoBRET signal, NanoBRET NanoGlo substrate (Promega) at a ratio of 1:166 to Opti-MEM media in combination with extracellular NanoLuc Inhibitor (Promega) diluted 1:500 (10 μL [30 mM stock] per 5 mL Opti-MEM plus substrate) were combined to create a 3X stock solution. A total of 50 μL of the 3X substrate/extracellular NanoLuc inhibitor were added to each well. The plates were read within 10 min on a GloMax Discover luminometer (Promega, Madison, WI, USA) equipped with 450 nm BP filter (donor) and 600 nm LP filter (acceptor), using 0.3 s integration time utilizing the “NanoBRET 618” protocol. Test compounds were evaluated at eleven concentrations in competition with NanoBRET Tracer K5 in HEK293 cells transiently expressing the AAK1 fusion protein. Raw milliBRET (mBRET) values were obtained by dividing the acceptor emission values (600 nm) by the donor emission values (450 nm) then multiplying by 1000. Averaged control values were used to represent complete inhibition (no tracer control: Opti-MEM + DMSO only) and no inhibition (tracer only control: no compound, Opti-MEM + DMSO + Tracer K5 only) and were plotted alongside the raw mBRET values. The data with n=3 biological replicates was first normalized and then fit using Sigmoidal, 4PL binding curve in Prism Software (version 8, GraphPad, La Jolla, CA, USA). All error bars are based on n=3 and are +/- standard error (SE). Flow cytometry: U2OS cells were treated with inhibitors at 5 μM, including controls (untreated and DMSO vehicle) and harvested 24 hours later. DNA synthesis detection. To detect DNA synthesis, cells were incubated with 10 µM 5-Ethynyl-2′-deoxyuridine (EdU, Santa Cruz Biotechnology) for 30 min before harvesting. Cells were harvested with 1X trypsin-EDTA (Sigma T4174) into 0.5 mL Eppendorf tubes and centrifuged at 2,150 xg for 3 min to remove trypsin. Following one wash with 500 µL cold 1X PBS, cells were centrifuged at 2,150 xg for 3 min again. After removing the supernatant, pellets were fixed with 500 µL 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 15 min at RT. 1 mL of 1% BSA/PBS was then added and mixed, and samples were centrifuged (2,150 xg for 5 min, the same centrifuge conditions were used for all subsequent flow cytometry steps). The supernatant was removed, cells were re-suspended in 1 mL 1% BSA/PBS and all samples kept at 4°C before labeling. EdU detection and DAPI staining. Cells were centrifuged, supernatants aspirated, and each sample resuspended in 1 mL 1% BSA/PBS + 0.5% Triton X-100 (Sigma T9284-100 mL) and incubated 15 min at RT. Cells were centrifuged again, supernatant aspirated, and cells incubated in PBS with 1 mM CuSO4,100 mM ascorbic acid in dH2O (freshly prepared right before use), and 1 µM Alexa Fluor 647–Azide (Life Technologies) for 30 min at RT in the dark. Cells were resuspended in 1 mL 1% BSA/PBS + 0.5% Triton X-100, and centrifuged. Finally, the supernatant was aspirated, and cells were incubated in 1 µg/ml DAPI (Sigma Aldrich) and 100 ng/ml RNase A (Sigma-Aldrich) in 1% BSA/PBS + 0.5% Triton X-100 overnight at 4°C in the dark. For negative control samples used to draw positive/negative gates, cells were not incubated with EdU but were labeled with Alexa Fluor 647–Azide but were labeled with DAPI. Data were acquired primarily on an Attune NxT flow cytometer (Thermo Fisher Scientific). Data were analyzed using FCS Express 6 (De Novo Software). Gating to separate debris from cells was determined using the forward scatter (FS) (area) versus side scatter (SS) (area) density dot

plots (“cells”: parent gate). Cells were then separated between singlets (desired) and doublets (eliminated) by gating on the plot of the DAPI (area) versus DAPI (height) density dot plots (parent gate: “cells” from FS vs. SS plot). The gate to isolate cell cycle phases was from DAPI area (DNA content) versus 647 area (DNA synthesis, EdU; parent gate: singlets). Each flow cytometry plot typically has 9,000–11,000 total single cells. CHEMISTRY Chemistry general procedures: Reagents were purchased from commercial suppliers and used without further purification. 1H and 13C NMR spectra were collected in methanol-d4 and recorded on Varian Inova 400 or Inova 500 spectrometers. Peak positions are given in parts per million (ppm); J values are expressed in hertz. Thin-layer chromatography (TLC) was performed on silica gel F254 to evaluate reaction courses and mixtures. Flash chromatography was performed on Merck 60 silica gel (0.063–0.200 mm). All non-aqueous reactions were performed under nitrogen atmosphere. Solutions containing the final products were dried over Na2SO4 before filtration and concentration under reduced pressure using a rotary evaporator. Additional characterization details for 14, 16 and 25 have been disclosed.19 N-(6-(3-((N,N-diethylsulfamoyl)amino)phenyl)-1H-indazol-3-yl)cyclopropanecarboxamide (25): 3-aminophenylboronic acid pinacol ester (1 eq., 50 mg, 0.228 mmol) in pyridine (1.2 mL) was cooled to 0oC in an ice bath. Diethylsulfamoyl chloride (2.06 eq., 80.7 mg, 0.0754 mL, 0.47 mmol) was added dropwise and the reaction mixture was stirred overnight. The reaction progress was checked by TLC, revealing complete consumption of SM. Solvent was removed and the residue was partitioned between dichloromethane and saturated aqueous sodium bicarbonate solution. The organic phase was dried (Na2SO4), filtered and concentrated. The residue was purified using an Isco Combiflash companion automated purification system (4g (18 mL/min), SiO2, 80%/20% to 25%/75% heptanes/EtOAc) to give diethyl({[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfamoyl})amine as an orange solid (44 mg, 54%), which was determined to be desired product by 1HNMR.

To a microwave vial was added N-(6-bromo-1H-indazol-3-yl)cyclopropanecarboxamide (1 eq., 20 mg, 0.0677 mmol), diethyl({[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfamoyl})amine (1 eq., 24 mg, 0.0677 mmol), and Pd(dppf)Cl2 (10%, 4.96 mg, 0.00677 mmol) in a mixture of dioxane (0.19 mL) and 1M aq sodium carbonate (0.16 mL). The vial was capped and heated in the microwave at 120oC for 30 min. Once cool, the reaction progress was examined by TLC, which revealed complete consumption of SM. The mixture was filtered through Celite and washed with EtOAc. The combined organic solution was washed with brine, dried (Na2SO4), filtered and concentrated. The residue was purified using an Isco Combiflash companion automated purification system (4g (18 mL/min), SiO2, 75%/25% to 20%/80% heptanes/EtOAc) to give 25 as a yellow solid (14.3 mg, 49%): 1H NMR (400 MHz, Methanol-d4) δ 7.80 (d, J = 0.8 Hz, 1H), 7.58 (s, 1H), 7.48 (dt, J = 0.8, 2.1 Hz, 1H), 7.38 – 7.35 (m, 2H), 7.33 (d, J = 8.5 Hz, 1H), 7.17 – 7.12 (m, 1H), 3.29 (d, J = 1.6 Hz, 4H), 1.91 (tt, J = 4.5, 8.0 Hz, 1H), 1.05 (t, J = 7.1 Hz, 6H), 1.01 (dt, J = 3.0, 4.4 Hz, 2H), 0.95 – 0.88 (m, 2H); 13C NMR (101 MHz, Methanol-d4) δ 174.1, 142.1, 141.9, 139.8, 139.0, 129.1, 122.2, 121.6, 119.7, 118.3, 118.1, 107.5, 48.2, 41.6, 13.5, 12.5, 6.8; HRMS: MS (ESI+): 427.17 (calcd), 427.12 (found).

N-(6-(3-((N-ethyl-N-methylsulfamoyl)amino)phenyl)-1H-indazol-3-yl)cyclopropanecarboxamide (26): 3-aminophenylboronic acid pinacol ester (1 eq., 50 mg, 0.228 mmol) in pyridine (1.2 mL) was cooled to 0oC in an ice bath. N-ethyl-N-methylsulfamoyl chloride (2.06 eq., 74.1 mg, 0.058 mL, 0.47 mmol) was added dropwise and the reaction mixture was stirred overnight. The reaction progress was checked by TLC, revealing complete consumption of SM. Solvent was removed and the residue was partitioned between dichloromethane and saturated aqueous sodium bicarbonate solution. The organic phase was dried (Na2SO4), filtered and concentrated. The residue was purified using an Isco Combiflash companion automated purification system (4g (18 mL/min), SiO2, 80%/20% to 25%/75% heptanes/EtOAc) to give ethyl(methyl){[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfamoyl}amine as a yellow solid (43 mg, 56%), which was determined to be desired product by 1HNMR.

To a microwave vial was added N-(6-bromo-1H-indazol-3-yl)cyclopropanecarboxamide (1 eq., 24 mg, 0.0857 mmol), ethyl(methyl){[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]sulfamoyl}amine (1 eq., 29.2 mg, 0.0857 mmol), and Pd(dppf)Cl2 (10%, 6.27 mg, 0.00857 mmol) in a mixture of dioxane (0.36 mL) and 1M aq sodium carbonate (0.30 mL). The vial was capped and heated in the microwave at 120oC for 30 min. Once cool, the reaction progress was examined by TLC, which revealed complete consumption of SM. The mixture was filtered through Celite and washed with EtOAc. The combined organic solution was washed with brine, dried (Na2SO4), filtered and concentrated. The residue was purified using an Isco Combiflash companion automated purification system (4g (18 mL/min), SiO2, 75%/25% to 20%/80% heptanes/EtOAc) to give 26 as a yellow solid (23 mg, 66%): 1H NMR (400 MHz, Methanol-d4) δ 7.83 (d, J = 8.5 Hz, 1H), 7.60 (s, 1H), 7.51-7.50 (m, 1H), 7.40 – 7.37 (m, 2H), 7.34 (d, J = 7.8 Hz, 1H), 7.21 – 7.16 (m, 1H), 3.24 (q, J = 7.2 Hz, 2H), 2.81 (s, 3H), 1.95- (tt, J = 4.5, 8.0 Hz, 1H), 1.28 (s, 1H), 1.08 (t, J = 7.1 Hz, 3H), 1.03 (m, 2H), 0.94-0.90 (m, 2H); 13C NMR (101 MHz, Methanol-d4) δ 174.1, 142.1, 141.9, 139.8, 138.9, 129.2, 122.2, 121.6, 119.7, 118.3, 118.1, 115.7, 107.5, 45.0, 33.1, 13.5, 11.8, 6.8; HRMS: MS (ESI+): 414.16 (calcd), 414.25 (found). N-(6-(3-((2-methylpropyl)sulfonamido)phenyl)-1H-indazol-3-yl)cyclopropanecarboxamide (27): 3-aminophenylboronic acid pinacol ester (1 eq., 50 mg, 0.228 mmol) in pyridine (1.2 mL) was cooled to 0oC in an ice bath. 2-methylpropane-1-sulfonyl chloride (2.06 eq., 73.6 mg, 0.0614 mL, 0.47 mmol) was added dropwise and the reaction mixture was stirred overnight. The reaction progress was checked by TLC, revealing complete consumption of SM. Solvent was removed and the residue was partitioned between dichloromethane and saturated aqueous sodium bicarbonate solution. The organic phase was dried (Na2SO4), filtered and concentrated. The residue was purified using an Isco Combiflash companion automated purification system (4g (18 mL/min), SiO2, 80%/20% to 25%/75% heptanes/EtOAc) to give 2-methyl-N-[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]propane-1-sulfonamide as a yellow solid (49 mg, 63%), which was determined to be desired product by 1HNMR.

To a microwave vial was added N-(6-bromo-1H-indazol-3-yl)cyclopropanecarboxamide (1 eq., 27 mg, 0.0964 mmol), 2-methyl-N-[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]propane-1-sulfonamide (1 eq., 32.7 mg, 0.0964 mmol), and Pd(dppf)Cl2 (10%, 7.05 mg, 0.00964 mmol) in a mixture of dioxane (0.49 mL) and 1M aq sodium carbonate (0.25 mL). The vial was capped and heated in the microwave at 120oC for 30 min. Once cool, the reaction progress

was examined by TLC, which revealed complete consumption of SM. The mixture was filtered through Celite and washed with EtOAc. The combined organic solution was washed with brine, dried (Na2SO4), filtered and concentrated. The residue was purified using an Isco Combiflash companion automated purification system (4g (18 mL/min), SiO2, 75%/25% to 20%/80% heptanes/EtOAc) to give 27 as a yellow solid (16 mg, 40%): 1H NMR (400 MHz, Methanol-d4) δ 7.83 (d, J = 8.6 Hz, 1H), 7.61 (s, 1H), 7.55 (d, J = 1.6 Hz, 1H), 7.45 – 7.41 (m, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.25 (dt, J = 2.1, 7.0 Hz, 1H), 3.01 (d, J = 6.3 Hz, 2H), 2.29-2.20 (m, 1H), 1.95-1.87 (m, 1H), 1.06 (d, J = 6.7 Hz, 6H), 1.03 (d, J = 4.4 Hz, 2H), 0.93 (dd, J = 3.5, 6.6 Hz, 2H); 13C NMR (101 MHz, Methanol-d4) δ 174.1, 142.4, 141.9, 139.9, 139.6, 138.7, 129.5, 122.9, 121.6, 119.7, 118.6, 118.5, 115.7, 107.6, 58.4, 24.6, 21.3, 13.5, 6.8; HRMS: MS (ESI+): 413.16 (calcd), 413.27 (found). N-(6-(3-(propylsulfonamido)phenyl)-1H-indazol-3-yl)cyclopropanecarboxamide (28): 3-aminophenylboronic acid pinacol ester (1 eq., 50 mg, 0.228 mmol) in pyridine (1.2 mL) was cooled to 0oC in an ice bath. Propane-1-sulfonyl chloride (2.06 eq., 67 mg, 0.0529 mL, 0.47 mmol) was added dropwise and the reaction mixture was stirred overnight. The reaction progress was checked by TLC, revealing complete consumption of SM. Solvent was removed and the residue was partitioned between dichloromethane and saturated aqueous sodium bicarbonate solution. The organic phase was dried (Na2SO4), filtered and concentrated. The residue was purified using an Isco Combiflash companion automated purification system (4g (18 mL/min), SiO2, 80%/20% to 25%/75% heptanes/EtOAc) to give N-[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]propane-1-sulfonamide as a red-orange solid (29 mg, 38%), which was determined to be desired product by 1HNMR.

To a microwave vial was added N-(6-bromo-1H-indazol-3-yl)cyclopropanecarboxamide (1 eq., 20.7 mg, 0.0738 mmol), N-[3-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]propane-1-sulfonamide (1 eq., 24 mg, 0.0738 mmol), and Pd(dppf)Cl2 (10%, 5.4 mg, 0.00738 mmol) in a mixture of dioxane (0.38 mL) and 1M aq sodium carbonate (0.19 mL). The vial was capped and heated in the microwave at 120oC for 30 min. Once cool, the reaction progress was examined by TLC, which revealed complete consumption of SM. The mixture was filtered through Celite and washed with EtOAc. The combined organic solution was washed with brine, dried (Na2SO4), filtered and concentrated. The residue was purified using an Isco Combiflash companion automated purification system (4g (18 mL/min), SiO2, 75%/25% to 20%/80% heptanes/EtOAc) to give 28 as a yellow solid (18 mg, 60%): 1H NMR (400 MHz, Methanol-d4) δ 7.83 (d, J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.55 (d, J = 1.7 Hz, 1H), 7.45 – 7.40 (m, 2H), 7.36 (d, J = 8.3 Hz, 1H), 7.25 (dt, J = 2.0, 7.1 Hz, 1H), 3.12 – 3.07 (m, 2H), 1.93 (tt, J = 4.5, 8.1 Hz, 1H), 1.86 – 1.78 (m, 2H), 1.04 – 0.98 (m, 5H), 0.93 (dd, J = 3.6, 7.6 Hz, 2H); 13C NMR (101 MHz, Methanol-d4) δ 174.1, 142.4, 141.9, 139.8, 139.6, 138.7, 129.5, 122.9, 121.6, 119.7, 118.6, 118.6, 107.6, 52.5, 16.9, 13.5, 11.6, 6.8; HRMS: MS (ESI+): 399.15 (calcd), 399.41 (found). REFERENCES 1. Derewenda, Z. S., Rational protein crystallization by mutational surface engineering. Structure 2004, 12 (4), 529-35. 2. Sorrell, F. J.; Szklarz, M.; Abdul Azeez, K. R.; Elkins, J. M.; Knapp, S., Family-wide Structural Analysis of Human Numb-Associated Protein Kinases. Structure 2016, 24 (3), 401-11.

3. Newman, J., Novel buffer systems for macromolecular crystallization. Acta crystallographica. Section D, Biological crystallography 2004, 60 (Pt 3), 610-2. 4. Chaikuad, A.; Knapp, S.; von Delft, F., Defined PEG smears as an alternative approach to enhance the search for crystallization conditions and crystal-quality improvement in reduced screens. Acta crystallographica. Section D, Biological crystallography 2015, 71 (Pt 8), 1627-39. 5. Kabsch, W., Xds. Acta crystallographica. Section D, Biological crystallography 2010, 66 (Pt 2), 125-32. 6. Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S., Overview of the CCP4 suite and current developments. Acta crystallographica. Section D, Biological crystallography 2011, 67 (Pt 4), 235-42. 7. McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J., Phaser crystallographic software. Journal of applied crystallography 2007, 40 (Pt 4), 658-674. 8. Cowtan, K., The Buccaneer software for automated model building. 1. Tracing protein chains. Acta crystallographica. Section D, Biological crystallography 2006, 62 (Pt 9), 1002-11. 9. Zhang, K. Y.; Cowtan, K.; Main, P., Combining constraints for electron-density modification. Methods in enzymology 1997, 277, 53-64. 10. Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K., Features and development of Coot. Acta crystallographica. Section D, Biological crystallography 2010, 66 (Pt 4), 486-501. 11. Chen, V. B.; Arendall, W. B., 3rd; Headd, J. J.; Keedy, D. A.; Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.; Richardson, D. C., MolProbity: all-atom structure validation for macromolecular crystallography. Acta crystallographica. Section D, Biological crystallography 2010, 66 (Pt 1), 12-21. 12. Schrödinger Release 2016-3: MacroModel, Schrödinger, LLC New York, NY, 2016. 13. Schrödinger Release 2016-4: Jaguar, Schrödinger, LLC, New York, NY, 2016. 14. Jester, B. W.; Cox, K. J.; Gaj, A.; Shomin, C. D.; Porter, J. R.; Ghosh, I., A coiled-coil enabled split-luciferase three-hybrid system: applied toward profiling inhibitors of protein kinases. J Am Chem Soc 2010, 132 (33), 11727-35. 15. Jester, B. W.; Gaj, A.; Shomin, C. D.; Cox, K. J.; Ghosh, I., Testing the promiscuity of commercial kinase inhibitors against the AGC kinase group using a split-luciferase screen. J Med Chem 2012, 55 (4), 1526-37. 16. Asquith, C. R. M.; Laitinen, T.; Bennett, J. M.; Godoi, P. H.; East, M. P.; Tizzard, G. J.; Graves, L. M.; Johnson, G. L.; Dornsife, R. E.; Wells, C. I.; Elkins, J. M.; Willson, T. M.; Zuercher, W. J., Identification and Optimization of 4-Anilinoquinolines as Inhibitors of Cyclin G Associated Kinase. ChemMedChem 2018, 13 (1), 48-66. 17. Davis, M. I.; Hunt, J. P.; Herrgard, S.; Ciceri, P.; Wodicka, L. M.; Pallares, G.; Hocker, M.; Treiber, D. K.; Zarrinkar, P. P., Comprehensive analysis of kinase inhibitor selectivity. Nature biotechnology 2011, 29 (11), 1046-51. 18. Vasta, J. D.; Corona, C. R.; Wilkinson, J.; Zimprich, C. A.; Hartnett, J. R.; Ingold, M. R.; Zimmerman, K.; Machleidt, T.; Kirkland, T. A.; Huwiler, K. G.; Ohana, R. F.; Slater, M.; Otto, P.; Cong, M.; Wells, C. I.; Berger, B. T.; Hanke, T.; Glas, C.; Ding, K.; Drewry, D. H.; Huber, K. V. M.; Willson, T. M.; Knapp, S.; Muller, S.; Meisenheimer, P. L.; Fan, F.; Wood, K. V.; Robers, M. B., Quantitative, Wide-Spectrum Kinase Profiling in Live Cells for Assessing the Effect of Cellular ATP on Target Engagement. Cell chemical biology 2017, 25 (2), 206-214.

19. Agajanian, M. J.; Walker, M. P.; Axtman, A. D.; Ruela-de-Sousa, R. R.; Serafin, D. S.; Rabinowitz, A. D.; Graham, D. M.; Ryan, M. B.; Tamir, T.; Nakamichi, Y.; Gammons, M. V.; Bennett, J. M.; Counago, R. M.; Drewry, D. H.; Elkins, J. M.; Gileadi, C.; Gildadi, O.; Godoi, P. H.; Kapadia, N.; Muller, S.; Santiago, A. S.; Sorrell, F. J.; Wells, C. I.; Fedorov, O.; Willson, T. M.; Zuercher, W. J.; Major, M. B., WNT activates the AAK1 kinase to promote clathrin-mediated endocytosis of LRP6 and establish a negative feedback loop. Cell reports 2019, 26 (1), 79-83.

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