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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 J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00164 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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Journal of Medicinal Chemistry

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,ς Taher Hegab, ⊥ David L. Cutler, ⊥ Jane W. S. Fang, ⊥ Gerald Fetterly, ⊥ Rudolf Kwan, ⊥ Allen Barnett, ⊥ Johnson Y. N. Lau, ⊥ David G. Hangauer,*⊥ ⊥

Athenex Inc., Conventus Building, 1001 Main Street, Suite 600, Buffalo, New York 14203

United States ς

Roswell Park Comprehensive Cancer Center, Department of Cancer Genetics & Genomics, Elm

and Carlton Streets, Buffalo, NY 14263 United States KEYWORDS: Src, Src inhibitor, tubulin, tubulin polymerization inhibitor, tyrosine kinase, protein tyrosine kinase, KX-01, KX2-391, KX01, KX-02, KX2-361, KX02, actinic keratosis, psoriasis, glioblastoma, GBM, anti-tumor agents, computer-aided-drug design, molecular modeling, rational drug design.

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

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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.

INTRODUCTION Src is a non-receptor protein tyrosine kinase (PTK), identified as the cellular proto-oncogene of the viral transforming gene, v-src, 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 (a.k.a. “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 to compete with mM intracellular concentrations of ATP.4,5a Some progress has been reported in discovering non-peptide small molecule Src inhibitors that are thought to target


2

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the peptide substrate binding site, however they generally are weak inhibitors, with IC50's >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.

RESULTS AND DISCUSSION Discovery Pathway for Clinical Src Inhibitors KX2-391 & KX2-361. The discovery pathway for the potent non-peptide, 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 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, non-peptide PTK


3


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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 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 non-peptide replacement for the dehydro-Phe residue. Finally, the position on the naphthalene scaffold that mimics the peptide substrate Tyr-OH was substituted with an α–hydroxymethyl 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 nonATP competitive manner, and was specific for PTKs over protein serine kinases.8 Compound 3 does not cross cell membranes, so a prodrug analog was prepared and evaluated in whole cells wherein a 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.


4

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HO

X

X=H, OH

N H

N O

H

IRTK peptide substrate

O

Dehydro-Phe analog (X=H)

OH HO

O P

X

HO

3

O

Non-peptide IRTK inhibitor with hybrid substrate-product IC50 = 95 M, Non-ATP competitive

Naphthalene conformationally restricted analog

Figure 2. Conceptual design process for 3 as a non-peptide, non-ATP competitive, insulin receptor tyrosine kinase inhibitor. 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 analogs. The pentapeptide Ac-Ile-Tyr-Gly-Glu-PheNH2, 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


5


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the substrate Tyr. An evaluation of the crystal structures of Src available at the time suggested the reason why.

Figure 3. The domain crystal structure of the inactive form of c-Src kinase (SH1) with nonhydrolyzable ATP analog, 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. One of the early crystal structures of Src was of the inactive form with a non-hydrolyzable ATP analog 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,


6

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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 side directed inhibitors when screening with isolated Src. Since Src binds to various multi-protein signaling complexes inside cells (e.g. EGFR, PDGFR, FAK, FGFR, etc) through its SH2 & 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, since 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 since 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


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would include multiple Src signaling complexes but this also raised the possibility that the 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 binding affinities did not improve significantly upon various peptide/peptide mimetic optimization efforts.


8

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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. 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 analog, 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 6 (GDYMNM) were resolved in the active site and the rest were too disordered to resolve (Figure 4). The auto-inhibited 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 non-peptide Src inhibitors that can penetrate whole cells (without requiring prodrug derivatization). This structure shows the auto-inhibitory IRTK Tyr residue 1,162 bound in the catalytic region of the active site, analogous to a peptide substrate Tyr. The Tyr 1,162 coordinates were used as a 3-dimensional template for a molecular modeling-based design of conformationally restricted Src inhibitor analogs with the assumption that this IRTK auto-inhibitory Tyr may bind in a similar conformation to a Src substrate Tyr. Building up on our earlier results with a naphthalene


9


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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 inhibitors16, 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 auto-inhibitory Tyr in 3-dimensional space with both the naphthalene and indole scaffolds. In order to gain initial confidence that these families of compounds are Src inhibitors, we tested an array of analogs against isolated Src.

Naphthalene scaffold analog 4 and indole scaffold analog 5 were

demonstrated to be non-ATP competitive inhibitors of isolated Src with IC50’s of 16 & 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 analog 5 was found to have anti-tumor activity with IC50’s of 4.2 µM, 59 µM 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.


10

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Figure 5.

Conceptual design for naphthalene and indole conformationally restricted Tyr

scaffolds and analogs 4 & 5 based upon IRTK auto-inhibited crystal structure. The three corresponding pharmacophore H-bonding groups are labeled 1 through 3 within the structures shown. 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). Since the IRTK peptide substrate binding site is partially formed, the Src peptide substrate binding site is also partially formed. Indole 5 was docked in the Src peptide substrate region of the active site as shown in Figure 6.


11


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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. In our indole scaffold SAR study16 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 di-flouro analog 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


12

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consequently replacing them with fluorine atoms as bioisosteric H-bond acceptors was also evaluated (Figure 7).16,18

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 Di-fluoro analog 6 was more potent that the di-hydroxy analog 5 against H460 cells, 15 µM 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 biaryl-based replacements19 for the indole side chain (Figure 7).


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We moved to testing our new analogs 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 auto-inhibitory 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 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 GI50’s of 0.41 µM, 1.03 µM, 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. In order to increase water solubility, a pyridyl nitrogen was substituted for CH at various positions in the 7 biaryl moiety19. These analogs indicated that the nitrogen position shown in 8 (Figure 7) provided the best potency, with GI50’s of 269 nM 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. In order 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


14

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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 (GI50’s 111 nM and 118 nM, respectively).19 We finally reconsidered the potential function of the meta-fluorine 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 remove the meta-fluorine from 9. Removing this fluorine improved the potency about 5-fold to 23 nM 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 analog 7 resulted in a large reduction in potency against the Src3T3 transformed cell line (5% inhibition at 1,000 nM).17


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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. The GI50 for 1 is 23 nM 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 nM and 6.5 nM, respectively, in HT29 and Src3T3 cells.20 Because most cells express multiple members of the 9-member Src-family kinases1, we addressed how 1 affects Src-specific proliferative signaling using mouse embryonic fibroblasts deleted of their Src, Fyn, and Yes


16

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genes(SYF cells) transduced with c-Src527F (c-Src527F/SYF). The GI50 for 1 against cSrc527F/SYF cells in the presence of 10% FCS and 2% FCS was 123 nM 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, since 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 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 al21 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 < 4 nM, Src3T3 cells IC50 110 nM).22 We utilized this ATP competitive Src inhibitor for some of our early comparative studies with 1.


17

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Change in Signal Intensity from Control


1.2

1.2

Src3T3

1

0.8

AZ28

0.6

1 391

IC50

0.6

IC50 0.4

0.2

0.2

GI50

GI50

0

Figure 9.

HT29

1

0.8

0.4

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0

C

0.2X

1X

5X

25X

C

Drug concentration x GI50

0.2X

1X

5X

25X

Drug concentration x GI50

GI50 (nM)

GI50 (nM)

AZ28 = 87 1 = 20

AZ28 = 647 1 = 25

Comparative inhibition of Src auto-phosphorylation (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 #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 auto-phosphorylation correlates with the growth inhibition for Src3T3 and HT29 cell lines, driven by active Src. Figure 9 shows the correlation between inhibition of Src auto-phosphorylation and cell proliferation for the Src growth driven cell lines Src3T3 and HT29. There is a good correlation for both compound 1 and the known ATP-competitive Src inhibitor AZ28, although compound 1 is more potent than AZ28 in both Src3T3 (20 nM vs. 87 nM, respectively) and HT29 cells (25 nM vs. 647 nM, respectively) in this comparative study. A further comparison in Src3T3 and HT29 cells showed a similar correlation between inhibition of transphosphorylation of the Src substrates Shc (Y239/240), FAK (Y925), and paxillin (Y-31) and growth inhibition.20 These results strongly suggest that compound 1 is a Src signaling inhibitor with ca. 20 nM level


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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 Supplemental Information). This 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&2, LCK, ZAP70, LYN and BCR-ABL auto-phosphorylation 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&2, LCK, and ZAP70 in whole cells up to a concentration of 10 µM.20 BCRABL and LYN were inhibited but at an IC50 of about 100-200 nM, i.e. about 10-fold 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 anti-proliferative effects.20 Compound 1 was then screened across a broad range of solid and liquid tumor cell lines as shown in Table 1. This data shows that 1 has broad anti-tumor activity in the low nM range, including against drug resistant tumor cell lines (see also Table 3). Compound 1 also showed a broader spectrum of activity, and greater potency, as compared to the ATP competitive Src inhibitor dasatinib, when evaluated against a series of liver cancer cell lines.33 Table 1. Potency of 1 and dasatinib against a range of solid and liquid tumor cell lines Human Solid Tumor Cell Line

1 GI50 (nM)

Human Liquid Tumor Cell Line

1 GI50 (nM)

dasatinib GI50 (nM)


19


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K562 (CML)

13

0.37

HT29 (Colon)

25

K562R (Gleevec resistant CML)

0.64

0.81

SKOV-3 (Ovarian)

10

MOLT-4 (ALL)

13

644

PC3-MM2 (Prostate)

9

CCRF-HSB-2 (ALL)

12

Inactive

L3.6pl (Pancreas)

25

Jurkat (Adult T cell leukemia)

10

8

MDA-MB-231 (Breast)

20

Ba/F3 + WT BCR-Abl

85

1

A549 (Lung)

9

Ba/F3 + E225K (Gleevec Resistant)

80

1

HuH7 (liver)

9

Ba/F3 + T315I (Gleevec & dasatinib Resistant)

35

>10,000

769-P (kidney)

45

KG-1 (AML)

16

Inactive

RPMI8226 (Multiple Myeloma)

40

Inactive

19

NT

RL (non-Hodgkin's lymphoma)

The oral bioavailability in mice for 1 (49%), wherein the fluorine was removed, was similar to that for 9 (64%) wherein the fluorine is present. The ability of 1 to penetrate the brain in mice was evaluated after oral administration at 10 mg/kg. The brain-to-plasma AUC ratio was 0.42 indicating the brain tissue exposure to 1 is 42% of the peripheral exposure. Although this is a respectable level of brain penetration for a small molecule oncology drug, a higher level would be more desirable for treating CNS cancers. The basicity of the morpholino nitrogen in 1 was expected to be a significant factor in affecting the ability to cross the blood-brain barrier (BBB) since the free base would likely be the form able to cross the lipophilic BBB. The number of rotatable bonds was also expected to be a significant factor. With these two factors in mind, analog 2 was designed to reduce the basicity of the morpholino nitrogen, due to conjugation with the aryl ring and attachment to a more electron withdrawing sp2 hybridized carbon atom, as well as a reduction in the number of rotatable bonds. The pKa of the morpholino nitrogen in 1 was measured at 6.16 (pyridyl N was 3.50) whereas the pKa of the morpholino nitrogen in 2 was 1.71 (pyridyl N was 3.66), showing that the basicity of 2 was greatly reduced to levels comparable


20

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with 1, hence yielding a much larger proportion of the corresponding free base at a physiological pH near 7.

Modeling studies with the homology built Src indicated that the shortened

morpholino side chain can be accommodated in the active site. The presence of a fluorine on the benzyl side chain was also postulated to increase the ability of the compound to cross the BBB. The brain-to-plasma AUC ratio for 2 in mice, after oral administration at 20 mg/kg, was 1.16 indicating that the brain tissue exposure is at least as high as the peripheral tissues.23 The oral bioavailability for 2 in mice was 40% when dosed at 20 mg/kg indicating that the dual goals of oral bioavailability and very high brain penetration were achieved with these targeted structural changes to 1.23 Compound 2 was demonstrated to be a Src auto-phosphorylation inhibitor in GL261 mouse glioblastoma cells with an IC50 of approximately 60 nM.23 Analogous to 1, 2 is a weak inhibitor of isolated Src with an IC50 of 67 µM, and likely for the same reasons. Brain penetrating 2 was tested against a range of CNS tumor cell lines as shown in Table 2, and found to have nM level potency against them all, including the Temodar-resistant cell line T98G.23 Table 2. Potency of 2 compared to dasatinib against a range of CNS tumor cell lines 2, GI50

dasatinib

(nM)

GI50 (nM)

Daoy

16

2,927

SK-N-MC

8

5,114

Neuroepithelioma (human)

SW1088

26

898

Astrocytoma (human)

LN-18

2.9

565

Glioma (human)

SK-N-FI

11

13

Neuroblastoma (human)

U87

76

1,586

Glioma (human)

GL261

57

18

Glioma (mouse)

Cell Line

Disease

Desmoplastic cerebellar medulloblastoma (human)


21


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T98G (Temodar

14

NT

Glioma (human)

U118

29

NT

Glioma (human)

U138

51

NT

Glioma (human)

U373

54

NT

Glioma (human)

resistant)

Discovery of Tubulin Polymerization Inhibition as the Second MOA for KX2-391 (1) & KX2-361 (2). The broad range of tumor cell lines in which compounds 1 and 2 showed potent growth inhibition suggested that a MOA in addition to Src inhibition may be involved for these compounds. This was reinforced by our comparative tumor cell studies with dasatinib/Sprycel (Tables 1, 2 & 3), a marketed ATP-competitive Src/multikinase inhibitor.24 Since Src will be inhibited by dasatinib, the finding that some cell lines showing sensitivity to 1 or 2 but not to dasatinib likely indicates that these cell lines are not Src-driven, again suggesting a second MOA for 1 and 2. Table 3. Tumor cell activity of 1 in dasatinib- and paclitaxel-resistant tumor cell lines Human Tumor Cell Line

1 GI50--GI90 (nM)

dasatinib GI50--GI90 (nM)

H460 (NSCLC))

51--162

90--48,880

H226 (NSCLC)

98--490

163--34,340

HCT116 (colon)

31--195

880--not reached

SW620 (colon)

109--903

2,418--2,940

1 GI50 (nM)

Paclitaxel GI50 (nM)

34

1,800

MEX-SA/Dx5 (Multi-drug resistant uterine sarcoma)


22

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NCI/ADR-RES (Multi-drug resistant ovarian cancer)

56

5,943

In order to identify the second MOA, we utilized photoaffinity labeling technology.25 We selected a benzophenone as the photoaffinity labeling moiety and an alkyne to subsequently attach a reporter tag with click chemistry after covalent attachment of the photoaffinity drug analog to the target proteins within whole cells as illustrated in Figure 10.


23


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Figure 10. Design of photoaffinity analog 10 from 1 and protein target(s) labeling in whole cells. Details of the labeling experiments are provided in the Supplementary Information. Importantly, photoaffinity analog 10 was equipotent to the parental drug 1 in both HT29 colon cancer cells (GI50’s 28 and 25 nM, respectively) and PC3-LN4 prostate cancer cells (GI50’s 45 and 40 nM, respectively) suggesting that the linkers did not alter drug targeting. HT29 colon cancer cells were treated with photoaffinity analog 10 in the presence and absence of a 200-fold excess of the parent drug 1 in order to identify possible off-target binding by the photoaffinity analog 10 (which should be lost in the presence of the parental drug). After photolabeling, lysates

were

treated

with

the

flourophore

TAMRA

azide

(Tetramethylrhodamine

azidetetramethylrhodamine 5-carboxamido-(6-azidohexanyl; Invitrogen B10182), which was appended through click chemistry with the alkyne, and then the labeled target proteins were separated by SDS-PAGE and visualized by in-gel fluorescent scanning (Figure 11).


24

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Figure 11. SDS-PAGE separation and in-gel fluorescent scanning visualization of protein targets labeled by photoaffinity analog 10 in the presence or absence of a 200-fold excess of the parental drug 1. Protein labeling that increased with higher concentrations of 10, and are lost in the presence of excess 1, are indicated by the arrows. These proteins appear to be targets for the parent drug 1. The left side of the figure indicates molecular weight markers in kD. One of the limitations of photoaffinity labeling technology is that the most abundant target proteins are the ones that are readily identified (Src abundance is likely too low to be identified by this experiment). As shown in Figure 11, a series of target proteins were labeled as potential drug target proteins. The protein at MW 55 kD was the most obvious drug target protein. A 2-D gel was then utilized to better separate the target proteins. The most strongly labeling proteins shown in Figure 12 were identified through in-gel digestion of excised bands, followed by UPLC Q-ToF MS/MS analysis. The labeled proteins were identified as α and β tubulin.26 With α and β tubulin identified as targets of photoaffinity analog 3 whose labeling can be competed with excess 1, the drug binding site and functional significance were explored. Figure 13 shows competition experiments of known tubulin binding drugs with photoaffinity analog 10 using 100- and 500-fold excesses of the listed drugs. These data clearly showed that only 1 can compete with photoaffinity analog 10, indicating that it binds to a novel site on tubulin.


25


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Figure 12. Analysis of labeled target proteins with 10 in HT29 cells by 2D gel. 1 mg of HT29 protein lysates were incubated with 2 µM 10 in the presence or absence of a 200-fold excess of the parent drug 1 on ice for 1h under UV irradiation at 365nm followed by click chemistry with a rhodamine-azide tag and 2D-gel electrophoresis. The gel spot indicated by the red circle was excised and subjected to in-gel trypsin digestion followed by LC-MS/MS (MALDI-TOF) analyses for protein identification. The protein IDs with high confidence score are listed in the accompanying table. Further labeling studies using 10 and purified tubulin along with mass spectroscopy analyses (Nano-LC/LTQ/OrbitrapETD) indicated that photoaffinity analog 10 (and parental drug 1) are likely binding at the interface of the α-β tubulin heterodimer.27 The functional significance of binding to the novel binding site on α-β tubulin heterodimer was first evaluated using a standard commercial in vitro tubulin polymerization assay kit (Cytoskeleton Inc, BK011P), which is based on the incorporation of a fluorescent reporter into microtubules as polymerization occurs, and showed that 1 is an effective tubulin polymerization inhibitor. The ability of 1 to disrupt the


26

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filamentous tubulin structures within tumor cells was then evaluated. As shown in Figure 14, compound 1 disrupts tubulin filaments in human peripheral blood mononuclear cells (PBMCs) in a dose-dependent fashion, with visible disruption evident starting at about 125 nM (about 6-fold higher than the Src inhibition IC50 in whole cells).

Figure 13. Competition of known tubulin-binding drugs with photoaffinity analog 10 (200 nM). Purified porcine brain tubulin (2 µg) was incubated with photoaffinity analog 10 on ice for 1h under UV irradiation at 365nm, followed by click chemistry with a TAMRA-azide tag, SDSPAGE separation and in-gel fluorescence scanning. The indicated inhibitors were included during the incubation at 100- or 500-fold molar excess relative to 10. The lack of competition by other known drugs indicates that 1 binds to a novel site on tubulin. The left side of the gel shows molecular weight markers in kD. When 1 is washed out of the cell culture, filamentous tubulin structures are restored within 30 minutes. In close agreement, an external laboratory’s study showed that inhibition of Src by 1 was evident at 20 nM in cell culture whereas tubulin polymerization inhibition required concentrations of at least 80 nM, i.e. at least 4-fold higher than Src inhibition.29b


27


Figure 14. Disruption of microtubule structures in PBMCs by 1.

Page 28 of 50

Human PBMCs were

treated with 1 at the indicated concentrations for 2 hours, followed by indirect immunofluorescence staining with anti-α-tubulin antibody. The same series of experiments were carried out with the brain-penetrating analog 2 in order to evaluate a second MOA. Compound 2 demonstrated competition with photoaffinity analog 10 for binding to purified tubulin in the same fashion as 1, and similarly 2 disrupted tubulin filaments in GL261 mouse glioma cells at 250 nM.23 This indicated that the Src and tubulin polymerization inhibition mechanisms are engaged for both 2 and 1. Compounds 2 and 1 also were found to cause G2/M cell cycle arrest, induction of mitotic catastrophe, increased apoptosis (enhanced cleavage of caspase 3 and PARP), increased aneuploidy, decreased cell migration, increased activation of acute stress-inducing molecules, and reduced p53 expression in tumor cell lines, in addition to Src inhibition in a number of external laboratories.23, 29, 30, 31


28

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A proteomics analysis of the effect of 1 and 2 on about 1,500 proteins in the PC3-LN4 prostate cancer cell line, as compared to the tubulin polymerization inhibitor vinblastine, was also carried out.28 The results showed that vinblastine, 1, and 2 all downregulated the expression of tubulin isotypes, however only 1 and 2 reduced autophosphorylated Src and affected protein expression levels in the Src signaling pathway. This analysis supported the dual Src and tubulin inhibition MOA for both 1 and 2. The impacts of 1 and 2 on these tumor cells, in regards to proteome changes, were found to be more similar to each other than either is to vinblastine, again supporting another MOA beyond tubulin polymerization inhibition.

Additionally, 1 and 2

affected proteomes slightly differently, suggesting overlapping yet unique subtilties in their effects on tumor cells. Importantly, it is also worth noting that 1 and 2 are not substrates for the efflux pump Pglycoprotein (P-gp) since 1 maintains its potency in the high Pgp-expressing paclitaxel-resistant cell lines shown in Table 3, and 2 is not excluded from penetrating the CNS due to the Pgp efflux pump active at the BBB.

Preclinical in vivo results with KX2-391 (1) & KX2-361 (2). In vivo anti-tumor activity of 1 was assessed using a broad range of orthotopic, transgenic, syngeneic and xenograft mouse solid and liquid tumor model studies. These include prostate (PC3-MM2-orthotopic and TRAMP C2H CaP-transgenic xenografts), breast (triplenegative MDA-MB-231 and MDA-MB-157, and estrogen receptor-α positive MCF7 orthotopic models), pancreatic (L3.6pl-orthotopic), colon (HT29-xenograft & CT26-syngenic), lung (NSCLC A549-xenograft), ovarian (mucinous xenografts RMUG-S and RMUG-L), and CML (BaF3[WT or BCR-Abl-xenograft]) models. Typical dosing in these mouse tumor models was


29


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oral either twice daily (5 mg/kg) or once daily (10-15 mg/kg) as a salt (HCl or mesylate), although additional dosing regimens are currently being evaluated. The half-life of 1 in mice after oral dosing is approximately 1 hour with an oral bioavailability of about 60%. In rats and dogs the half-life of 1 is approximately 2 and 8 hours, respectively, and it is also well absorbed orally (50-90%). The twice daily dosing in mouse models was selected when only the Src inhibition MOA was known. Based upon our discovery of tubulin polymerization inhibition as the second MOA, once daily dosing was evaluated in order to achieve a higher Cmax and thereby better engage the less potent tubulin MOA as well. All of the mouse model studies demonstrated good efficacy of 1 against tumor growth. An analysis of the tumor vs. plasma AUC in an HT29 xenograft mouse model after oral dosing at 5 mg/kg gave a ratio of 1.52, showing an enhanced tumor tissue exposure relative to the plasma levels.

The mouse model studies evaluating 1 against triple negative breast cancer29,

ERα positive breast cancer30, and mucinous ovarian cancer31 have been published. In these external lab studies, in vivo inhibition of tubulin and Src was demonstrated in isolated tumor tissues, as well as in tumor cell cultures. This resulted in inhibition of tumor growth, reduced tumor cell proliferation, increased apoptosis, anti-angiogenesis, and importantly, reduced metastasis. Synergistic in vivo and in vitro efficacy of 1 with tamoxifen for estrogen receptor-α (ERα) positive breast cancer30, paclitaxel or doxorubicin for triple negative breast cancer29a, and oxaliplatin for mucinous ovarian cancer31 was also demonstrated.

Compound 1 was more

efficacious in inhibiting metastasis than was paclitaxel29a, even though both compounds disrupt tubulin dynamics, albeit by inhibition of tubulin polymerization for 1 vs. inhibition of tubulin depolymerization for paclitaxel. This suggests that Src inhibition as the second MOA for 1 may be increasing its efficacy in blocking metastasis since Src activity has been linked to metastasis.2


30

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Interestingly, reducing the oral dose of 1 to 20% of the full dose (i.e. to 1 mg/kg twice daily) in triple negative orthotopic mouse breast cancer models caused re-expression of ERα through changes in histone acetylation, along with increases in the levels of a series of ERα targets, and a mesenchymal-to-epithelial transition.32 These changes resulted in a re-sensitization to tamoxifen treatment in the mouse models. The re-expression of ERα in these models did not occur when 1 was administered at the full dose (5 mg/kg twice daily) wherein tubulin disruption is also observed. On the other hand, Src inhibition was observed at the lower dose, suggesting that Src inhibition is related to the ERα re-expression. This effect was reversible so that continuous daily dosing with 1 was needed to maintain ERα re-expression and sensitization to tamoxifen. These results suggested that triple negative breast cancer patients could potentially have less tumor burden and reduced metastasis from daily oral low dose administration of 1 in addition to cotreatment with tamoxifen and/or other endocrine therapy agents.

This drug combination

treatment might provide a low toxicity alternative to current cytotoxic chemotherapy cocktails used to treat these breast cancer patients. Compound 2 was also evaluated in mouse tumor models.

Compound 2 has an oral

bioavailability of about 40%, a half-life of about 2.5 hours, and excellent brain penetration (as mentioned earlier) in mice.

Most relevant for compound 2 is a stereotactic intracerebral

implantation GL261 glioblastoma multiforme (GBM) tumor model in syngeneic mice. This model is thought to provide a good simulation for GBM in patients because the mouse GL261 tumors tend to grow into the surrounding brain tissues in mice with immune systems. Compound 2, dosed orally at 30 mg/kg once daily, produced about 30% long term survivors (i.e. > 100 days) whereas vehicle produced 0% survivors within < 25 days.23 This effect was found to require a fully functional immune system since repeating the experiment in SCID mice lacking T


31


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and B cells resulted in longer survival but not >75 days. Additionally, re-challenging 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 re-dose 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 anti-tumor 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) & 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 hour period. In the initial trial design the continuous BID dosing was carried out for 3 weeks and the 4th week was a drug holiday in each four week cycle. The maximum tolerated dose in this late stage cancer patient population was determined to be 40 mg per dose BID with the 3 weeks on, 1 week off dosing schedule. Subsequently, a continuous dosing schedule without the 4th week off was evaluated and the 40 mg per dose BID was still


32

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well tolerated resulting in this as the final maximum tolerated dose (MTD) and dosing schedule. The most common dose-limiting-toxicities were liver function test abnormalities (i.e. increased AST and ALT but without increased bilirubin), that were reversible within one week, and fatigue. Compound 1 was well absorbed orally and the drug exposures were dose-proportional in these patients. At the MTD 40 mg dose the half-life was approximately 4 hours, the Tmax occurred at about 1 hour, and the Cmax was about 80 ng/ml (186 nM). The apparent volume of distribution (Vz/F) was about 681 L indicating good tissue penetration.

The highest dose

administered in this clinical study was 80 mg/dose BID resulting in a Cmax of 273 ng/ml (633 nM). There was no evidence of drug accumulation over repeated days of dosing. Compound 1 showed preliminary suggestions of potential efficacy in some of the Phase I patients. An example is a patient with metastatic ovarian cancer as shown in Fig. 15. This patient had been on nine prior therapy regimens, including tubulin targeting drugs. The longest duration of prior therapy was 5 months (carboplatin-paclitaxel combination). This patient’s CA125 ovarian cancer biomarker clearly responded to compound 1 treatment, and the patient remained on the treatment for 16 months in spite of the extensive prior drug therapies. This patient achieved a Cmax of 231 ng/ml (536 nM) in the first monthly cycle with a 50 mg/dose BID, significantly higher than the Cmax mean of 80 ng/ml at the MTD of 40 mg/dose BID.


33


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Figure 15. Phase I metastatic ovarian cancer patient sequence of prior therapy before compound 1 (left side), and response of patient CA-125 ovarian cancer biomarker to compound 1 (right side). Compound 1 completed a Phase 2 clinical study in bone-metastatic castration-resistant prostate cancer patients (CRPC), the results of which have been published.35 This clinical study also commenced prior to the tubulin polymerization inhibition second MOA being discovered for compound 1. Consequently, the trial was conducted with repeated twice daily oral dosing of compound 1 at the Phase 1 MTD, i.e. 40 mg/dose BID. The median Cmax for this dosing regimen was 61 ng/ml (142 nM), lower than the 80 ng/ml Cmax for the MTD 40 mg dosing in the Phase 1 study with advanced solid tumor patients. This dose of compound 1 was well tolerated in the Phase 2 study and adverse events were generally mild to moderate. The Phase 2 trial did not meet the efficacy endpoints, 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


34

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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 anti-tumor 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 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. Since 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 hours. 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 cm to 4 cm and survived 373 days. An example of a shorter duration, higher dose, is patient that was treated


35


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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 Supplemental 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 consideration and pre-clinical 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 intra-epithelial 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 Since 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


36

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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.


37


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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 non-peptide analogs, 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 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 hour 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


38

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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 pre-cancerous 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


39


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and clinical applications can be better designed once the full MOA, and potency for each, is known along with the PK. Supporting Information Information related to the chemical and structural formulas, including molecular formula strings, 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, is provided in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author *E-mail: [email protected]. Tel.: 716-573-3654. ACKNOWLEDGEMENTS We gratefully thank Dr. Latif Kazim and his coworker Kyoung-Soo Choi, Roswell Park Comprehensive Cancer Center, for Mass Spectrum data identifying tubulin as the major protein to which photoaffinity analog 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. ABBREVIATIONS 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, cSrc527F/NIH3T3 engineered cell line expressing a constitutively-active, oncogenic Src; SYF


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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 cSrc527F; BBB, blood-brain-barrier; PBMCs, peripheral blood mononuclear cells; MTD, maximum tolerated dose; BID, twice daily dosing; AK, actinic keratosis; SCC, squamous cell carcinoma. REFERENCES (1) (a) Martin, G. S. The hunting of the Src. Nature Reviews Molecular Cell Biology 2001, 2, 469-475. (b) Martin, G. S. The road to Src. Oncogene, 2004, 23, 7910–7917. (2) (a) Frame, M.C. Src in cancer: Deregulation and consequences for cell behavior. Biochim. Biophys. Acta, 2002, 1602, 114-130. (b) Frame, M.C. Newest findings on the oldest oncogene; how activated src does it. Journal of Cell Science, 2004, 117, 989-998. (c) Summy, J. M.; Galllick, G. E. Src family kinases in tumor progression and metastasis. Cancer and Metastasis Reviews, 2003, 22, 337-358. (d) Summy, J. M.; Gallick, G. E. Treatment for advanced tumors: Src reclaims center stage. Clinical Cancer Research, 2006, 12(5), 1398-1401. (e) Yeatman, T. J. A renaissance for Src. Nature Reviews Cancer, 2004, 4, 470-480. (f) Kim, M. P.; Park, S. I.; Kopetz, S.; Gallick, G. E. Src family kinases as mediators of endothelial permeability: effects on inflammation and metastasis. Cell Tissue Research, 2009, 335(1), 249-259. (g) Kopetz, S.; Shah, A. N.; Gallick, G. E. Src continues aging: current and future clinical directions. Clinical Cancer Research, 2007, 13(24), 7232-7236. (3) Zhang, S.; Dihua Yu, D. Targeting Src family kinases in anti-cancer therapies: turning promise into triumph. Trends in Pharmacological Sciences, 2012, 33, No. 3, 122-128.


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Zafer, K.; Ali Riza, G. Efficacy and safety assessment of 0.5% and 1% colchicine cream in the treatment of actinic keratosis. Journal of Dermatological Treatment, 2001, 12(4), 199203. (39) Siegel, J. A.; Korgavkar, K.; Weinstock, M. A. Current perspective on actinic keratosis: a review. British Journal of Dermatology, 2017, 177, 350–358. (40) Compound 1 clinical trials for actinic keratosis listed on clinicaltrials.gov: (a) Phase 1; NCT02337205. A phase 1, single-center, safety, tolerability, and pharmacokinetic study of KX2 391 ointment in subjects with actinic keratosis. (b) Phase 2; NCT02838628. A phase 2a, open-label, multicenter, activity and safety study of KX2-391 ointment in subjects with actinic keratosis on the face or scalp. (c) Phase 3; NCT03285477. 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. (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. 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. Journal of cutaneous pathology, 2008, 35(3), 273-277.


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(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. Table of Contents Graphic


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Discovery of Novel Dual Mechanism of Action Src ... - ACS Publications

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