Discovery of Novel Dual Mechanism of Action Src Signaling and

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Drug Annotation Cite This: J. Med. Chem. 2018, 61, 4704−4719

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Discovery of Novel Dual Mechanism of Action Src Signaling and Tubulin Polymerization Inhibitors (KX2-391 and KX2-361) Michael P. Smolinski,† Yahao Bu,† James Clements,† Irwin H. Gelman,‡ Taher Hegab,† David L. Cutler,† Jane W. S. Fang,† Gerald Fetterly,† Rudolf Kwan,† Allen Barnett,† Johnson Y. N. Lau,† and David G. Hangauer*,† †

Athenex Inc., Conventus Building, 1001 Main Street, Suite 600, Buffalo, New York 14203, United States Department of Cancer Genetics & Genomics, Roswell Park Comprehensive Cancer Center, Elm and Carlton Streets, Buffalo, New York 14263, United States

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S Supporting Information *

ABSTRACT: The discovery of potent, peptide site directed, tyrosine kinase inhibitors has remained an elusive goal. Herein we describe the discovery of two such clinical candidates that inhibit the tyrosine kinase Src. Compound 1 is a phase 3 clinical trial candidate that is likely to provide a first in class topical treatment for actinic keratosis (AK) with good efficacy and dramatically less toxicity compared to existing standard therapy. Compound 2 is a phase 1 clinical trial candidate that is likely to provide a first in class treatment of malignant glioblastoma and induces 30% long-term complete tumor remission in animal models. The discovery strategy for these compounds iteratively utilized molecular modeling, along with the synthesis and testing of increasingly elaborated proof of concept compounds, until the final clinical candidates were arrived at. This was followed with mechanism of action (MOA) studies that revealed tubulin polymerization inhibition as the second MOA.

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to compete with mM intracellular concentrations of ATP.4,5a Some progress has been reported in discovering nonpeptide small molecule Src inhibitors that are thought to target the peptide substrate binding site, however they generally are weak inhibitors, with IC50s > 10 μM against isolated Src and in whole cell assays.5 The goal of the drug discovery program described herein was to develop first in class clinically relevant, orally available, Src inhibitors that target the peptide substrate site at nM potencies and that have high selectivity among kinases.

INTRODUCTION Src is a nonreceptor protein tyrosine kinase (PTK), identified as the cellular proto-oncogene of the viral transforming gene, vsrc, encoded by Rous sarcoma virus.1 Elevated levels of Src activity have been shown to play an important role in primary tumor growth and, perhaps even more significantly, in metastasis.2 The initial promise of Src as an oncology drug target has resulted in a number of ATP-competitive Src inhibitors progressing into clinical trials, however, none has shown strong efficacy as a monotherapy against solid tumors.3 A confounding factor is the homology among tyrosine kinase ATP binding sites, thereby resulting in significant cross activity against a range of additional kinases (aka “multikinase” inhibitors) for ATP site directed inhibitors. The potential advantages of targeting the more unique kinase peptide substrate sites with small molecule inhibitors has been recognized by researchers for decades although this goal has proven generally challenging to achieve.4,5a Potential advantages include much higher kinase inhibition selectivity and greater efficacy within cells because the inhibitor will not need © 2018 American Chemical Society

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RESULTS AND DISCUSSION Discovery Pathway for Clinical Src Inhibitors KX2-391 and KX2-361. The discovery pathway for the potent nonpeptide, non-ATP competitive, oral Src inhibitors 1 (KX2-391, KX-01, KX01)6 and 2 (KX2-361, KX-02, KX02)7 shown in Figure 1 traces back almost 30 years to the initial Received: January 31, 2018 Published: April 4, 2018 4704

DOI: 10.1021/acs.jmedchem.8b00164 J. Med. Chem. 2018, 61, 4704−4719

Journal of Medicinal Chemistry

Drug Annotation

IC50 of 10 μM was found, demonstrating that the mM intracellular levels of ATP did not affect potency.8 In light of the simplicity of 3, and its potency in whole cells, this early compound encouraged us to continue down a similar conceptual path for Src. The next step was to identify a small peptide substrate for Src that could be used as a basis for designing initial modified peptide inhibitor analogues. The pentapeptide Ac-Ile-Tyr-GlyGlu-Phe-NH2 was identified as the best known small peptide Src substrate with a Km of 368 μM when tested with isolated Src.9 A general synthetic approach to mimetics of this pentapeptide, containing unnatural amino acid replacements for tyrosine, was developed, including an α-hydroxymethyl phosphonate moiety replacement for a substrate tyrosine OH.10 It was found that small peptides bound weakly to isolated Src, even with unnatural amino acid replacements for the substrate Tyr. An evaluation of the crystal structures of Src available at the time suggested the reason why. One of the early crystal structures of Src was of the inactive form with a nonhydrolyzable ATP analogue bound in the ATP substrate site (Figure 3).11 This structure showed a well-formed ATP substrate binding site along with an open, poorly formed, peptide substrate binding site. Subsequent Src crystal structures reflecting active forms also did not show a well-formed peptide substrate binding cavity and were not solved with a peptide inhibitor in the active site.12 Overall, the Src crystal structures suggested that the ATP substrate site is generally well formed, whereas the peptide substrate site is not, at least when Src is separated from its intracellular protein binding partners. This likely explains why tight binding of ATP site directed inhibitors are much more readily identified than peptide site directed inhibitors when screening with isolated Src. Because Src binds to various multiprotein signaling complexes inside cells (e.g., EGFR, PDGFR, FAK, FGFR, etc.) through its SH2 and SH3 domains, we considered the possibility that these Src protein−protein interactions within the various signaling complexes could allosterically cause the Src peptide substrate binding cavity to be fully formed. Furthermore, because each of these dynamic signaling complexes would have its own constellation of proteins for potentially affecting the Src conformation, the shape of the Src peptide substrate binding cavity might differ somewhat from one complex to the next, thereby possibly reducing “cross talk” among the various Src signaling complexes. This would offer the potential for extending the selectivity beyond Src vs other PTKs to Src within certain signaling complexes over others. The Src peptide substrate binding cavity could also potentially be altered as protein members of the signaling complex bind or dissociate as well as when their post translational modification states change. This may result in a large number of Src substrates across multiple signaling complexes, an observation that has been made with anti-poTyr blots of Src-transformed cells.13 However, these novel concepts presented a practical challenge in that the kinase inhibitor testing methodologies common in the field at the time did not readily apply because various reconstituted multiprotein signaling complexes would need to be utilized. The isolated kinase inhibition assays would not be expected to show potent Src inhibition because the peptide substrate binding site is poorly formed so that the inhibitor would need to capture a rare conformation of the isolated protein, a kinetically and thermodynamically unfavorable process. Whole cell assays would include multiple Src signaling complexes, but this also raised the possibility that the

conceptual approach described in the 1989 publication from Hangauer and colleagues, then at Merck.8

Figure 1. Structure of dual MOA Src and tubulin polymerization inhibitors that are under clinical evaluation.

In this early proof-of-concept study, the insulin receptor tyrosine kinase (IRTK) was used as a model PTK to determine the feasibility of rationally designing small molecule, nonpeptide, PTK inhibitors that target the peptide substrate binding site. The flow of the conceptual genesis for the IRTK inhibitor 3 is outlined in Figure 2 and described in more detail

Figure 2. Conceptual design process for 3 as a nonpeptide, non-ATP competitive, insulin receptor tyrosine kinase inhibitor.

in the 1989 publication.8 The basic design concept was to start with the tyrosine residue that is phosphorylated in peptide substrates, taking note of the discovery that a dehydro-Phe can be substituted for Tyr in peptide Src inhibitors, and then utilize a naphthalene scaffold as a conformationally restricted nonpeptide replacement for the dehydro-Phe residue. Finally, the position on the naphthalene scaffold that mimics the peptide substrate Tyr-OH was substituted with an αhydroxymethyl phosphonate moiety as a hybrid substrate(OH)/product(phosphate) mimetic for interacting with the IRTK catalytic residues. Compound 3, albeit a simple compound, inhibited IRTK peptide substrate transphosphorylation with a Ki of 95 μM in a non-ATP competitive manner and was specific for PTKs over protein serine kinases.8 Compound 3 does not cross cell membranes, so a prodrug analogue was prepared and evaluated in whole cells wherein an 4705



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Figure 3. Domain crystal structure of the inactive form of c-Src kinase (SH1) with nonhydrolyzable ATP analogue, AMP−PNP (red) bound in the ATP site (PDB 2SRC).11 The structure is displayed as a Molcad surface, and the generated image was colored based on hydrophobic potential using Sybyl 6.9.

Figure 4. Color coded MOLCAD substrate site surface of the active form of the insulin receptor tyrosine kinase (PDB 1IR3)14a with AMP−PNP bound (left side) and the visible region (GDYMNM, right side) of the peptide substrate KKKLPATGDYMNMSPVGD shown as atom color-coded ball and stick structures. Brown and green surfaces are hydrophobic and blue surfaces are hydrophilic as generated using Sybyl 6.9. The substrate tyrosine is clearly seen adjacent to the ATP mimic terminal phosphate. Hydrogen bonds are shown as dashed lines.

detection of Src inhibition by standard Western blots, interrogated by Src auto- and transphosphorylation-specific antibodies, may be dependent upon the particular cell line used as well as the status of the various Src signaling complexes in response to the cell growth conditions used. Nevertheless, transitioning the peptide site directed Src inhibitor discovery

efforts from isolated Src to whole cells wherein the Src multiprotein signaling complexes are formed seemed to be needed. This conclusion was based upon the structural information available showing the poorly formed Src peptide substrate binding site, the μM level binding affinities of peptide substrates and inhibitors with isolated Src, and our finding that 4706



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Figure 5. Conceptual design for naphthalene and indole conformationally restricted Tyr scaffolds and analogues 4 and 5 based upon IRTK autoinhibited crystal structure. The three corresponding pharmacophore H-bonding groups are labeled 1 through 3 within the structures shown.

Figure 6. Docked indole Src inhibitor 5 in the Src peptide substrate site built upon a shared homology to that in IRTK. The Src structure is displayed as a Molcad surface, and the generated image was colored based on hydrophobic potential using Sybyl 6.9. H-bonds are indicated by yellow dotted lines, and some of the key interactions are indicated.

binding affinities did not improve significantly upon various peptide/peptide mimetic optimization efforts. While transitioning to whole-cell Src inhibition testing, we still needed qualitative structural guidance as to the families of compounds likely to bind to the Src peptide substrate site. The only PTK crystal structures available at the time that were solved with a peptide bound in the at least a partially formed peptide substrate binding site were for the insulin receptor tyrosine kinase (IRTK).14 The activated IRTK structure was solved at 1.9 A resolution with the nonhydrolyzable ATP analogue, adenylyl imido-diphosphate (AMP-PNP), bound in

the ATP substrate site, and the 18-residue substrate, KKKLPATGDYMNMSPVGD (Km 24 μM), bound in the peptide substrate site.14a Of these 18 amino acids, only six (GDYMNM) were resolved in the active site and the rest were too disordered to resolve (Figure 4). The autoinhibited IRTK complex14b was published earlier and was available at the time of our initial efforts to use it as a qualitative guide for the design of nonpeptide Src inhibitors that can penetrate whole cells (without requiring prodrug derivatization). This structure shows the autoinhibitory IRTK Tyr residue 1,162 bound in the catalytic region of the active 4707



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Figure 7. Conceptual genesis of clinical compound 1 from indole Src inhibitor 5. Inhibition data for compounds 6 through 1 have been reported previously.19

docked in the Src peptide substrate region of the active site as shown in Figure 6. In our indole scaffold SAR study,16 we noted that the position of the benzyl amide OH in 5 contributed more significantly to potency than the position of the indole OH. This observation stimulated us to consider a reversed binding mode, i.e., one wherein the benzyl amide OH is positioned analogous to a Tyr substrate OH and the indole scaffold serves as the side chain. This reverse binding mode for 5, and the corresponding difluoro analogue 6, were successfully modeled in the Src active site built upon homology to the IRTK active site. We also noted that the two hydroxyl groups in 5 are expected to be potential sites of rapid phase II metabolism in vivo, and consequently replacing them with fluorine atoms as bioisosteric H-bond acceptors was also evaluated (Figure 7).16,18 Difluoro analogue 6 was more potent than the dihydroxy analogue 5 against H460 cells, 15 vs 59 μM, respectively.17 This result, along with the two key insights that the Src binding mode might be reversed and that the OH’s can be replaced with less metabolically labile F’s, encouraged us to proceed further down this design pathway to the clinical compound 1 as outlined in Figure 7. We then addressed whether the indole side chain could be replaced with a different side chain in order to enhance potency. A number of possibilities were modeled using the Src homology built structure. We settled on a biarylbased replacements19 for the indole side chain (Figure 7). We moved to testing our new analogues against the HT29 highly active Src colon cancer cell line and a c-Src527F/ NIH3T3 engineered cell line (“Src3T3” in Figure 7), expressing a constitutively active, oncogenic Src (due to replacement of the autoinhibitory Tyr 527 with Phe). Src3T3 cells were grown with a low initial seeding density, low growth factor serum levels (1.5% FCS), and harvested well short of confluence in order to maximize their dependence on Src signaling for cell growth. Compounds that show potent proliferation inhibition under these conditions would be considered likely Src signaling inhibitors. Under low serum conditions, the GI50 of 7 was 349

site, analogous to a peptide substrate Tyr. The Tyr 1,162 coordinates were used as a 3-dimensional template for a structured-based design (SBD) of conformationally restricted Src inhibitor analogues with the assumption that this IRTK autoinhibitory Tyr may bind in a similar conformation to a Src substrate Tyr. Building up on our earlier results with a naphthalene scaffold-based, non-ATP competitive, IRTK inhibitor described above, we applied this scaffold to the design of conformationally restricted Src inhibitors.15 The same approach was used to design analogous indole-based scaffold Src inhibitors,16 both of which are illustrated in Figure 5. Comparative molecular modeling studies indicated that the key hydrogen bonding moieties labeled 1, 2, and 3 in Figure 5 can be positioned analogously to the autoinhibitory Tyr in 3dimensional space with both the naphthalene and indole scaffolds. To gain initial confidence that these families of compounds are Src inhibitors, we tested an array of analogues against isolated Src. Naphthalene scaffold analogue 4 and indole scaffold analogue 5 were demonstrated to be non-ATP competitive inhibitors of isolated Src with IC50s of 16 and 38 μM, respectively.15,16 Also the structure−activity relationships with these scaffolds15,16 confirmed that the H-bond moiety positions shown in Figure 5 are the optimal locations. Indole analogue 5 was found to have antitumor activity with IC50s of 4.2, 59, and 41 μM against KM12 (colon), H460 (lung, NSCL), and A549 (lung, NSCL) tumor cell lines, respectively, all of which have active Src.17 Comparing these tumor cell potencies to the isolated Src IC50 of 38 μM demonstrates that the potency does not decrease when tested against whole cells, suggesting no significant effects due to high intracellular ATP levels or cell penetration limitations. Encouraged by these initial results from qualitative modeling, the IRTK ternary complex (IRTK:AMP−PNP:GDYMNM)14a was then used to construct a homology model of an analogous Src complex with the peptide inhibitor removed (see Supporting Information). Because the IRTK peptide substrate binding site is partially formed, the homology built Src peptide substrate binding site is also partially formed. Indole 5 was 4708



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Figure 8. Docked clinical Src inhibitor 1 in the Src homology-built peptide substrate site. The Src structure is displayed as a Molcad surface, and the generated image was colored based on hydrophobic potential using Sybyl 6.9. H-Bonds are indicated by yellow dotted lines, and some of the key interactions are indicated.

remove the meta-fluorine from 9. Removing this fluorine improved the potency about 5-fold to 23 and 13 nM against HT29 and Src3T3 cells, respectively.19 Our model for 1 binding in the homology built Src peptide substrate binding site is shown in Figure 8. Figure 8 shows the 1 biaryl pyridyl nitrogen atom engages in a hydrogen bond with Src and the morpholinoethoxy group extending down the peptide substrate binding channel. The amide NH that connects the benzyl moiety to the biaryl moiety is also engaged in a hydrogen bond with Src. This modeled hydrogen bond with Src is in agreement with our finding that N-methylating the amide NH in the earlier analogue 7 resulted in a large reduction in potency against the Src3T3 transformed cell line (5% inhibition at 1000 nM).17 The GI50s for 1 are 23 and 13 nM against HT29 and Src3T3 cell lines, respectively, when grown in the presence of our standard low growth factor concentration (1.5% FCS). When the growth factor concentration was reduced from 1.5% to 0.5% FCS, thereby further increasing the dependency on Src signaling for growth as described earlier, the potency was improved to 5 and 6.5 nM, respectively, in HT29 and Src3T3 cells.20 Because most cells express multiple members of the nine-member Src-family kinases,1 we addressed how 1 affects Src-specific proliferative signaling using mouse embryonic fibroblasts deleted of their Src, Fyn, and Yes genes (SYF cells) and transduced with c-Src527F (c-Src527F/SYF). The GI50s for 1 against c-Src527F/SYF cells in the presence of 10% FCS and 2% FCS were 123 and 23 nM, respectively.20 The 5-fold improvement in potency when the growth factor concentration is decreased strongly suggests that 1 is a Src signaling inhibitor because other PTKs would become more involved in controlling growth at the higher growth factor concentrations, leading to decreased potency of a selective Src signaling inhibitor. The inhibition of isolated Src by 1 was weak (IC50 46 μM), as was expected based upon our earlier Src inhibition results described above including the structural observations that the

nM, suggesting that this compound is a Src signaling inhibitor in whole cells. Wider testing of 7 against KM12 (colon), A549 (NSCLC), and HT29 (colon) cancer cell lines provided GI50s of 0.41, 1.03, and 0.88 μM, respectively.19 These results showed that the biaryl side chain had broken the micromolar potency barrier when tested in whole cells. The next task was to increase the water solubility so that oral bioavailability could be obtained and to further increase the potency. To increase water solubility, a pyridyl nitrogen was substituted for CH at various positions in the 7 biaryl moiety.19 These analogues indicated that the nitrogen position shown in 8 (Figure 7) provided the best potency, with GI50s of 269 and 338 nM against the Src3T3 and HT29 tumor cell lines, respectively.19 Modeling studies with homology built Src indicated that an aryl nitrogen at that position may function as a H-bond acceptor with Src. Unfortunately, the water solubility increase was not enough to provide good oral bioavailability, which was only 4% for 8 in mice. To further increase the water solubility, a morpholinoethoxy side chain was appended to 8 to produce 9 (Figure 7). Modeling studies of the complex for 9 with homology built Src indicated that the para position of the terminal phenyl ring would be an acceptable position for this water solubilizing side chain because it would allow the morpholinoethoxy side chain to further extend down the peptide substrate binding channel or extend out into water. Adding this side chain not only increased the oral bioavailability in mice to 64% but also increased the potency against HT29 and Src3T3 cells by about 3-fold (GI50s 111 and 118 nM, respectively).19 We finally reconsidered the potential function of the metafluorine in 9. We initially postulated that this fluorine might enhance binding by functioning as a hydrogen bond acceptor for the Src catalytic Arg-388, similar to a substrate OH, but were also concerned that this interaction may be negated by a repulsive interaction with the nearby catalytic Asp-386 carboxylate. Consequently, the final structural modification that provided the clinical compound 1 (Figure 7) was to 4709



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Figure 9. Comparative inhibition of Src autophosphorylation (Src-poY416) vs growth inhibition for Src3T3 and HT29 cells. Compound 1 is compared to the ATP competitive Src inhibitor AZ28.22 Cells were lysed, subjected to PAGE, and probed with phospho-Src family (poTyr416) antibody (Cell Signaling Technology no. 2101). Densitometry was used to quantify the results from the gels and the Y axis shows the fold change in the Src-Y416 densitometry signal from the control (no inhibitor). The X axis is scored with multiples of the individual compound’s GI50. The activity against Src autophosphorylation correlates with the growth inhibition for Src3T3 and HT29 cell lines, driven by active Src.

Table 1. Potency of 1 and Dasatinib against a Range of Solid and Liquid Tumor Cell Lines human solid tumor cell line HT29 (colon) SKOV-3 (ovarian) PC3-MM2 (prostate) L3.6pl (pancreas) MDA-MB-231 (breast) A549 (lung) HuH7 (liver) 769-P (kidney)

1 GI50 (nM) 25 10 9 25 20 9 9 45

human liquid tumor cell line

1 GI50 (nM)

dasatinib GI50 (nM)

K562 (CML) K562R (Gleevec resistant CML) MOLT-4 (ALL) CCRF-HSB-2 (ALL) Jurkat (adult T cell leukemia) Ba/F3 + WT BCR-Abl Ba/F3 + E225 K (Gleevec resistant) Ba/F3 + T315I (Gleevec and dasatinib resistant) KG-1 (AML) RPMI8226 (multiple myeloma) RL (non-Hodgkin’s lymphoma)

13 0.64 13 12 10 85 80 35 16 40 19

0.37 0.81 644 inactive 8 1 1 >10000 inactive inactive NT

potency in whole cells. These results were confirmed by Src kinase inhibition data, in addition to inhibition of downstream signals, in various tumor cell lines and tumor tissues carried out by a number of external laboratories.23,29a,b,30,31 The selectivity of Src inhibitor 1 compared to AZ28 across a range of isolated kinases was tested at a set concentration of 10 μM (see Supporting Information). These data showed that 1 has no activity against the 16 kinases tested, whereas AZ28 showed strong cross inhibitory activity against ABL, EGFR, FYN, LCK, LYN, and YES. The ability of 1 to inhibit Pdgfr, Egfr, Jak1 and Jak2, LCK, ZAP70, LYN, and BCR-ABL autophosphorylation in Src/Yes/Fyn-null (SYF) mouse embryo fibroblasts, MDA-MB-231 (LCK), Jurkat (ZAP70), DU-145 (LYN), and K562 (BCR-ABL) cell lines was also evaluated. These results demonstrated that 1 does not inhibit Pdgfr, Egfr, Jak1 and Jak2, LCK, and ZAP70 in whole cells up to a concentration of 10 μM.20 BCR-ABL and LYN were inhibited, but at an IC50 of about 100−200 nM, i.e., about 10fold less potently than against Src.20 Compound 1 also induced p53 expression (a tumor suppressor) in Src3T3 cells, PARP cleavage (an apoptosis indicator) in K562 cells and caspase-3 cleavage (an apoptosis indicator) in Src3T3 and HT29 cells, suggesting several mechanisms for its antiproliferative effects.20 Compound 1

peptide substrate site is not well formed when Src is isolated. Indeed, using 1 as a control to develop an NMR-based assay with a paramagnetic probe for the identification of non-ATP competitive kinase inhibitors, Moy et al.21 confirmed that 1 binds to isolated Src outside of the ATP site, simultaneously with an ATP-site binder, suggesting that it binds in the peptide substrate site. This contrasts with Ple et al., who reported an ATP competitive anilinoquinazoline Src inhibitor (compound 28 in their publication, labeled AZ28 herein) with high affinity and specificity for Src (isolated Src IC50 100 days), whereas vehicle produced 0% survivors within 75 days. Additionally, rechallenging syngeneic mice that had survived, due to prior dosing with 2, with subcutaneous or intracerebral GL216 reimplantation, resulted in rejection of the tumors in all of the mice without needing to redose 2. Immuno-histochemical analysis of the mice from the original experiment that did grow tumors revealed the presence of CD3+ and CD8+ T cell infiltrates into the tumor tissues.23 A cohort of mice that survived >75 days were followed to the end of their normal life expectancy (ca. 2 years), and tumor recurrence was not observed in any of the mice. Taken together, these data indicated that compound 2 combines two avenues of antitumor efficacy, a Src/tubulin targeted effect and an engagement of an adaptive immune response to the glioblastoma tumor cells. This suggests that the potential utility of combining compound 2 with immunotherapy agents for the treatment of glioblastoma may also be a fruitful future avenue to pursue. Clinical Evaluation of KX2-391 (1) and KX2-361 (2). Compound 1 has completed a phase 1 clinical trial in solid tumor patients, and the results have been published.34 This clinical trial was completed before the second MOA (tubulin) for 1 was discovered and consequently before once daily dosing was considered to be better able to engage the tubulin MOA. Consequently, the trial evaluated continuous twice daily (BID) oral dosing based upon the early mouse BID dosing model studies in order to increase the Src inhibition coverage over a 24 h period. In the initial trial design, the continuous BID 4715



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consideration and preclinical evaluation. Oral dosing provides the flexibility to evaluate such dosing regimens (including “metronomic” dosing) in order to optimize the efficacy vs toxicity profile. A number of literature reports have linked Src activity to the premalignant intraepithelial lesion actinic keratosis (AK) and squamous cell carcinoma (SCC).37 About 65% of SCC’s arise from AK lesions.37a,b Tubulin polymerization inhibition has also been reported to be effective in treating AK lesions.38 Because compound 1 has both MOAs, this opened up another clinical development opportunity. Compound 1 inhibits the growth of human keratinocytes in vitro with an IC50 of 32 nM. AK is one of the most common skin conditions that dermatologists see in their patients and is considered a major health concern due to its ability to progress into keratinocyte carcinomas such as SCC.39 A topical formulation for compound 1 was developed and evaluated in phase 140a and phase 240b clinical trials and has proceeded into two phase 340c,d clinical trials for AK. The phase 2 study41 demonstrated that the compound 1 ointment, at a 1% strength and administered for only 5 consecutive days to the AK field, was well tolerated. The 100% clearance of AK lesions from the field was 52% on the face and 33% on the scalp.41 This preliminary efficacy, along with the mild local skin reactions that resolved rapidly, formed the basis for proceeding into two phase 3 trials. The combination of good efficacy with low local skin toxicity sets compound 1 apart from the leading current field treatments for AK wherein good efficacy is coupled with significant local skin toxicity, and promises to provide a new, first in class, treatment for this common precancerous skin disease. The much lower skin toxicity of 1, particularly, is expected to result in wider patient and dermatologist acceptance for AK field treatments. Psoriasis is also a hyperproliferative skin disease and compound 1 is a potent inhibitor of human keratinocyte proliferation. Additionally, Src activity has been shown to be elevated in psoriasis as well as other hyperproliferative skin diseases such as AK and SCC.42 Consequently, topical formulations of compound 1 are also being evaluated in an ongoing phase 1 psoriasis clinical trial.43 Compound 2 has proceeded into phase 1 clinical trials with oral administration in solid tumor patients.44 The primary objective is to define the MTD. Secondary objectives are to determine safety and tolerability, PK, and preliminary efficacy. The phase 1 results are not yet available to be released. However, the exceptional brain penetration of 2, coupled with the 30% long-term survivors in the mouse GBM model, suggest that 2 could provide a badly needed first in class efficacious therapy for GBM going forward.

and adverse events were generally mild to moderate. The phase 2 trial did not meet the efficacy end points, however, a modest effect on bone metabolism markers and circulating tumor cell (CTC) counts was observed. The tubulin polymerization inhibition second MOA was discovered after this trial had concluded. A retrospective analysis of the plasma levels of compound 1 likely needed to begin to engage the tubulin second MOA in solid tumor patients indicated that levels above 142 ng/mL (329 nM) would be needed and can be achieved by dosing at 80 mg/dose or above based upon the phase 1 PK data.35 This analysis included the measured effect of plasma binding in cell culture experiments and the 52% enhanced concentration of compound 1 in preclinical mouse tumor tissues mentioned earlier. It was concluded that the 40 mg/ dose BID dosing did not take full advantage of the antitumor activity of compound 1 and that once daily dosing, at a significantly higher dose, should be evaluated in future clinical studies. In order to evaluate once daily dosing, and to diversify the clinical studies into liquid tumors, a phase 1b study with compound 1 was carried out in advanced elderly acute myeloid leukemia (AML) patients.36 A MTD of about 120 mg/dose, repeated once daily, was established in this patient population. Once daily dosing, even in these frail patients, allowed the administered dose to be increased 3-fold and the total daily dose to be increased by 50%. Compound 1 partitioned into the bone marrow at concentrations similar to the plasma concentrations. Because there is no enhanced concentration of 1 in the bone marrow, unlike solid tumor tissue, then the threshold for beginning to engage the tubulin MOA in these liquid tumor patients is about 216 ng/mL (500 nM). At the 120 mg MTD dose, the median Cmax was 218 ng/mL (506 nM), with a half-life of about 2.2 h. This drug level is significantly above the 142 ng/mL needed for solid tumor patients, assuming enhanced compound 1 concentrations in the patient’s tumor tissues as was observed in preclinical studies, but is just reaching the desired 216 ng/mL range for liquid tumor patients. Higher single daily doses for shorter durations, with drug free days in between, are suggested for liquid tumor patients by these PK results. Nevertheless, indications of some efficacy were observed in this phase 1b AML trial. For example, one patient treated with 120 mg repeated daily doses for 165 days had a reduction in splenomegaly from 16 to 4 cm and survived 373 days. An example of a shorter duration, higher dose is a patient that was treated with a 160 mg dose repeated daily for only 12 days and then remained treatment free for about 18 months. The combined phase 1 and 2 PK results for compound 1 administered orally in solid and liquid tumor patients, along with the concurrent discovery of the tubulin polymerization inhibition second MOA, were very instructive for determining how this compound will likely need to be dosed to maximize its efficacy vs toxicity profile. Peripheral neuropathy, a common side effect of tubulin targeting drugs such as paclitaxel (depolymerization inhibitor) and vincristine (polymerization inhibitor), was not caused by compound 1 in the clinical trials to date. This reduced clinical neurotoxicity is supported by the observation that 1 did not alter neurite morphology in a neurite outgrowth assay, whereas vincristine showed a strong effect (see Supporting Information). Other dosing regimens, such as higher single daily doses for a small number of consecutive days, with an appropriate number of drug-free days between the contiguous dosing days, (e.g., “pulse” dosing) are under

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CONCLUSION The drug discovery journey described herein was a long one, spanning almost 30 years, albeit with significant blocks of time wherein the project was dormant. It started with the discovery of pentapeptide Src substrate/inhibitors along with some simple naphthalene and indole based small molecule Src (and IRTK) inhibitors that were designed to target the peptide substrate site. The weak binding affinity of peptide substrates/ inhibitors, and early nonpeptide analogues, to isolated Src, the poorly formed peptide substrate site in the Src crystal structures, and the knowledge that Src functions in cells while bound within various multiprotein signaling complexes, led to the conclusion that the inhibitors needed to be tested in 4716



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a whole cell environment. This was a critical insight for the project. Cell growth experiments, wherein the growth is highly dependent upon Src activity, were used to test increasingly elaborated Src inhibitors within a whole cell environment wherein Src is bound to growth controlling signaling complexes. The iterative use of structural/modeling information and proof of concept compounds, followed by potency and in vivo PK optimization, led to two clinical compounds, 1 and 2. Initial mouse xenograft and early clinical trials with 1 were designed to provide more Src inhibition coverage in a 24 h cycle, and hence twice daily dosing was used. Later MOA studies identified tubulin polymerization inhibition as a second, but somewhat less potent, MOA. An estimation of the plasma levels needed to significantly engage the tubulin second MOA indicated that once daily dosing, at a significantly higher dose, was needed to take advantage of the two MOAs in one drug activity. This was the second important insight in the project. Other dosing regimen variations are also possible to optimize the time at which the plasma levels meet or exceed what is required to engage the tubulin MOA. Dosing in mice verified that the once daily dose can be about 3-fold higher than one of the twice daily doses and that efficacy can be achieved. The MTD dose in oncology patients was also increased from 40 mg/dose twice daily to 120 mg/dose once daily, mirroring the mouse 3-fold increase results. While oral clinical trials with compounds 1 and 2 in oncology patients were underway, the idea of evaluating a topical formulation of 1 for hyperproliferative skin diseases such as actinic keratosis (AK) and psoriasis arose. Phase 1 and 2 clinical trials with 1 for AK were very promising, and pivotal phase 3 registration clinical trials are currently underway. The unprecedented combination of good efficacy with low local skin toxicity for field AK treatment is providing a breakthrough for treating this common precancerous skin disease. Although the AK clinical application was not envisioned as a potential indication when this program was initiated, it has accelerated quickly. This was the third important insight in the drug discovery and development project. A phase 1 clinical trial of 1 for psoriasis has also been initiated. Fundamental medicinal chemistry insights were applied to compound 1 in order to greatly enhance the ability to cross the blood−brain barrier, resulting in compound 2. Compound 2 is a rare example of a potent small molecule oncology compound that fully penetrates the brain. In addition to having a dual Src/ tubulin polymerization inhibition MOA, this compound also engages the immune system in a mouse syngeneic model of GBM to further enhance its efficacy and produce long-term survivors. Adding this triple efficacy MOA spectrum to the oral bioavailability, and facile brain penetration, makes compound 2 a first in class and highly promising compound for treating GBM and other CNS malignancies. Some of the more general take away concepts from this drug discovery journey are that protein targets that bind within multiprotein complexes in cells may need to be tested in their natural whole cell environment. When doing so, other MOAs may also be unknowingly incorporated into the compound which require subsequent MOA studies to identify. The dosing and clinical applications can be better designed once the full MOA, and potency for each, is known along with the PK.

Drug Annotation

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00164. Chemical and structural formulas, and the synthesis of compounds 1, 2, 7, 8, 9, and 10, pKa determinations of compounds 1 and 2, homology modeling, in vitro biological testing, pharmacokinetic studies, and in vivo tumor drug distribution (PDF) Molecular formula strings (CSV) SrcH1 + compound 1 (PDB) SrcH1 + indole 5 (PDB)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 716-573-3654. E-mail: [email protected]. ORCID

David G. Hangauer: 0000-0002-0497-369X Notes

The authors declare the following competing financial interest(s): Michael P. Smolinski, Yahao Bu, James Clements, David L. Cutler, Jane W. S. Fang, Gerald Fetterly, Rudolf Kwan, Allen Barnett, Johnson Y. N. Lau, and David G. Hangauer are all either employed by Athenex, Inc. and/or hold equity in Athenex, Inc.

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ACKNOWLEDGMENTS We gratefully thank Dr. Latif Kazim and his co-worker KyoungSoo Choi, Roswell Park Comprehensive Cancer Center, for mass spectrum data identifying tubulin as the major protein to which photoaffinity analogue 10 covalently binds (Figure 12), and Dr. Jun Qu, The University at Buffalo, for mass spectrum data identifying the interface between α−β tubulin heterodimer as the likely binding site for 10.

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ABBREVIATIONS USED MOA, mechanism of action; AK, actinic keratosis; GBM, glioblastoma multiforme; PTK, protein tyrosine kinase; Src, pp60c‑src; ATP, adenosine triphosphate; IRTK, insulin receptor tyrosine kinase; EGFR, epidermal growth factor receptor; PDGFR, platelet derived growth factor receptor; FAK, focal adhesion kinase; FGFR, fibroblast growth factor receptor; SH2, Src homology 2 domain; SH3, Src homology 3 domain; FCS, fetal calf serum; Src3T3, c-Src527F/NIH3T3 engineered cell line expressing a constitutively active, oncogenic Src; SYF cells, mouse embryonic fibroblasts deleted of their Src, Fyn, and Yes genes; c-Src527F/SYF, mouse embryonic fibroblasts deleted of their Src, Fyn, and Yes genes and transduced with c-Src527F; BBB, blood−brain barrier; PBMCs, peripheral blood mononuclear cells; MTD, maximum tolerated dose; BID, twice daily dosing; AK, actinic keratosis; SCC, squamous cell carcinoma; SBD, structure-based design

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REFERENCES

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safety study of KX2-391 ointment in subjects with actinic keratosis on the face or scalp.. (c) Phase 3: NCT03285477. A phase 3, doubleblind, vehicle-controlled, randomized, parallel group, multicenter, efficacy, and safety study of KX2−391 ointment 1% in adult subjects with actinic keratosis on the face or scalp.. (d) Phase 3: NCT03285490. A phase 3, double-blind, vehicle-controlled, randomized, parallel group, multicenter, efficacy and safety study of KX2-391 ointment 1% in adult subjects with actinic keratosis on the face or scalp.. (41) Fang, J.; Jarratt, M.; Kempers, S.; Forman, S.; Cutler, D.; Wang, H.; Kwan, R. Phase II study of KX2-391 ointment 1%, a novel field treatment based on Src/tubulin polymerization inhibition, for actinic keratosis. In American Academy of Dermatology Meeting in Feb. 17, 2018. Oral presentation and online ePoster ID 6134. (42) Ayli, E. E.; Li, W.; Brown, T. T.; Witkiewicz, A.; Elenitsas, R.; Seykora, J. T. Activation of Src-family tyrosine kinases in hyperproliferative epidermal disorders. J. Cutaneous Pathol. 2008, 35 (3), 273−277. (43) Taiwan clinical trial KX01-PS-01-TW in collaboration with Athenex Inc. partner PharmaEssentia Corp. (44) ClinicalTrials.gov, NCT02326441, A phase 1 clinical study to evaluate the safety, tolerability and pharmacokinetics of KX2-361 in subjects with advanced malignancies that are refractory to conventional therapies.

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Discovery of Novel Dual Mechanism of Action Src Signaling and

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