A strategy for the design of multiplex inhibitors for kinase-mediated signalling in angiogenesis

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A strategy for the design of multiplex inhibitors for kinase-mediated signalling in angiogenesis Jerry Adams*, Pearl Huang and Denis Patrick Tumour growth is dependent on multiple factors, including the physiological process of angiogenesis. Several opportunities for inhibiting angiogenesis with targeted therapies have been identified and are currently being evaluated for clinical efficacy. Some of the most promising approaches include smallmolecule inhibitors for the tyrosine receptor kinase VEGFR2. Other signal-transduction pathways have also been shown to regulate angiogenesis, including FGFR, PDGFR, Tie and EphB. Addresses GlaxoSmithKline MMPD CEDD Departments Oncology and Medicinal Chemistry, Upper Merion, King of Prussia, Philadelphia 19406, USA *e-mail: [email protected] Current Opinion in Chemical Biology 2002, 6:486–492 1367-5931/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations EGFR epidermal growth factor receptor FGFR fibroblast growth factor receptor PDGFR platelet-derived growth factor receptor RTK receptor-linked tyrosine kinase VEGFR vascular endothelial growth factor receptor

Introduction The recruitment and maintenance of vasculature to the environment of the tumour cell is complex and is regulated by multiple cell-signalling pathways. Endothelial growth and survival are affected by multiple growth factors and cytokines. The receptors for vascular endothelial growth factor (VEGFR2), angiopoetins (Tiel-2), platelet-derived growth factors (PDGFR1–2), B ephrins (EphB2 and EphB4) and the fibroblast growth factors (FGFR1–4) are receptor-linked tyrosine kinases (RTKs) that stimulate endothelial proliferation in a tumour environment. Each of these receptor kinases is worth consideration as a specific target for inhibition and small-molecule inhibitors are being aggressively pursued for several of these kinases, most notably VEGFR2 [1–3]. Several groups have demonstrated that tumours are capable of secreting multiple angiogenic growth factors [4,5]. This realization and the marginal efficacy achieved in some tumour models in which a single angiogenic pathway is blocked have led researchers to speculate that compounds which simultaneously inhibit multiple kinase pathways (‘multiplex inhibitors’) may be required to achieve maximal clinical efficacy [6,7]. The feasibility of identifying suitable multiplex inhibitors has now been demonstrated with the entry of several such inhibitors into clinical trials. Following a brief review of the current lead RTK inhibitors, this manuscript presents a proposal to explain the observation of multiplex-inhibition and

the pharmacological data that supports the continued investigation of these agents.

The selectivity of angiogenic kinase inhibitors Table 1 lists RTK inhibitors targeted at blocking angiogenesis along with their tyrosine kinase selectivity, their development status and company name (for structures, see Figure 1). With the exception of the Tie2 inhibitor BSF 466895, the primary targets of these compounds are the pro-angiogenic kinases VEGFR2, PDGFRβ and FGFR1. Using a definition of more than 10-fold difference in IC50 between the potency for inhibition of the primary and secondary targets as the definition of selectivity as applied to the data in Table 1, examples of selectivity inhibitors are BSF 466895, ZD-6474 and SU-6688. However, for each of these examples, a closer examination reveals that none of these compounds fully meet this definition. In the case of BSF 466895, the IC50 for inhibition of Tie1 is only ninefold greater than that of Tie2 [8]. For ZD-6474 and SU-6688, the selectivity determined for inhibition of the activity of the isolated kinase domain did not ensure pharmacological selectivity. In addition to blocking VEGFR2, ZD-6474 is effective in vivo at blocking EGFR (epidermal growth factor receptor) signalling [9,10]. The ~300-fold difference in PDGFR and VEGFR2/KDR potency for SU-6688 disappears in cell-based assays where similar IC50 values for inhibition of receptor autophosphorylation were determined for both receptors [11••]. Clearly, the definition of selectivity used above is inadequate. Firstly, a tenfold window does not ensure target specificity. A more useful definition may be a 100-fold difference, but as cited above for SU-6688 this may also prove inadequate. Secondly, for the examples where data are available, it appears that selectivity within an RTK family is rarely achieved (see Table 1 and Figure 2). Hence, we propose that for these RTKs it is useful to apply the discussion of selectivity across and not within families. Using a 100-fold difference in IC50 and looking at cross-family selectivity, the only compound that would fit this second definition of selectivity is the Tie2/Tie1 kinase inhibitor BSF 466895. Although the available data are incomplete, the trend suggested in Table 1 is that these inhibitors target TRK classes 3 and 4 and, to a lesser extent, class 5. In general, Class 1 and 2 RTKs, as well as the non-receptor Src tyrosine kinases, are weakly inhibited. Moreover, none of these agents are potent inhibitors of serine/threonine kinases, which comprise >80% of the kinases in the human protein kinase family (data not shown in table).

Structural rationale The sequencing of the human genome has now enabled a systematic classification of the entire kinase family, which

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Table 1 Selectivity of angiogenic kinase inhibitors. Kinase class Compound PTK787 Novartis ZD-6474 AstraZeneca SU-5416 Sugen SU-6668 Sugen SU-11248 Sugen CP-547,632 Pfizer CEP-7055 Cephalon AG013736 Pfizer PD-173074 Pfizer PD-166285 Pfizer KRN633 Kirin CHIR200131 Chiron BSF 466895 Abbott

RTK 1

RTK 2

Status

EGFR

IRK or IGF-1R

RTK 3

RTK 4

RTK 5

RTK 10

N-RTK

PDGFRb

KIT

c-fms VEGFR1 VEGFR2 VEGFR3

FGFR

Tie2

Src

Phase II

>10000

ND

580

730

1400

77

37

640

Phase I

500

>20000

1100

ND

ND

1600

40

ND

3600

2500

ND

[9,10]

Discont >100000 ND -inued Phase I/II >100000 >10000

320†

100*

ND

ND

160†

ND

19500†

ND

ND

[38]

8†

ND

ND

ND

2100†

ND

1200†

ND

>10000

[11••]

>10000 >10000 >10000

Refer -ences [37]

Phase I

>100000

ND

8

1–10

ND

ND

9

ND

830*

ND

ND

[39]

Phase I

6000

11000

870

ND

ND

ND

11

ND

9

ND

ND

[40]

Phase I

>1000

25000

460

ND

ND

12

4–14

17

162

>10000

ND

[41]

Phase I

ND

ND

ND

ND

ND

2.6†

3.7†

ND

56†

~1000

>1000

[7]

>50000

17.6

ND

ND

ND

100-200*

ND

45.2†

ND

20000

[25]

>50000

139†

ND

ND

ND

ND

ND

54.4†

ND

4.9†

[42]

Preclinical >10000

>10000*

130*

9.6*

190*

12*

1.2*

ND

>10000*

ND

>10000

[43]

Preclinical 50–500

ND

51

7

12

13

8

ND

ND

[33,34]

7890

790

8720

2670

830

680

5

1060

[8]

Preclinical >50000 Preclinical

Preclinical

105†

28800

50-500 ND ND

ND

Kinases are subgrouped into families based on genomic structure [24,44]. All data reported as IC50 values for kinase inhibition in cell-free systems except as noted; ND denotes no data. *Cellular IC50. †Ki determination. Cell color code: green, IC50 < 100 nM; yellow, IC50 between 100 and 5000 nM; red, IC50 > 5000 nM.

is estimated at around 500 proteins for humans [12••]. With the determination of significant numbers of protein kinase crystal structures, it is now clear that the homology that was apparent from the primary sequence gives rise to a single family of tertiary structures in which the catalytic domains and ATP-binding site in the active conformation of these kinases are remarkably similar. By contrast, there is a remarkable diversity of structure for these same kinases in their unactivated state. The regulation of this conformational plasticity is believed to be a key feature in the regulation of kinase signaling [13,14]. An understanding of these regulatory mechanisms also promises to be key to developing selective inhibitors. The idea that inhibitor selectivity might segregate with kinase family homology is intuitive. However, there are many examples of kinase inhibitors whose selectivity profile is not restricted to closely related families [15••]. This has in turn led to the common belief that kinase inhibitor selectivity is unpredictable. The data presented in this review (Table 1) suggests, at least for some classes of kinases and inhibitors, the pattern of selectivity can be predicted with reasonable confidence. Our proposal for why this might be so follows.

Although there are selected examples of protein kinase inhibitors that do not bind in the ATP site, these are clearly the exceptions [16]. Based upon the available precedent from X-ray structural data and enzyme kinetics, it is reasonable to assume that all the kinase inhibitors presented in Table 1 bind in the ATP pocket. In those cases in which structural data are available, the known ATP competitive inhibitors mimic in some manner the backbone hydrogen bond donor–acceptor interaction that is used to engage N-1 and N-6 in the adenine base of ATP [17]. The utilization of this common ATP-binding pocket was initially viewed as a fatal shortcoming of ATP competitive inhibitors that would lead to pan-active kinase inhibitors. However, this has not proven to be true. Many structurally diverse and highly selective ATP-binding pocket inhibitors have been described [16,18]. For the design of multiplex inhibitors, the utilization of the ATP-binding site, if not a requirement, is highly desirable as it provides the possibility of a common and predictable binding site across the kinase targets of interest. Assuming that the hydrogen bond donor–acceptor site used by ATP affords a common site for inhibitor binding, one can assign the features that give rise to inhibitor

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Figure 1

H 3C

F

X

Br

HN N

CH 3

O

N H

N

HN

N

O

N

Cl

N

N

O

N

HN N

O

Cl

Cl

N

SU-5416, X = H SU-6668, X = CH2CO2H

ZD-6474

PTK787

N

PD-166285

O N

Br H N

F O

O

NH 2 O

O

F

O

O

N

N

N H

S

N H

HN

N O Cl

HN

O

O

CEP-7055 Pro-drug to CEP-5214

Cl

S

NH

N

N

HN O

CP-547,632 O

O

N

N

N

PD-173074

F Cl

NH 2 N

NH2 HN

N N

N

N N H

BSF 466895

O

CHIR200131

N

N

N

H

O

O O O

H N

H N

N N

KRN633

Current Opinion in Chemical Biology

Structures of angiogenic kinase inhibitors.

selectivity into two general categories, local and global. Local influences are those changes in specific protein residues in or near the ATP-binding pocket that directly impact the shape and or electrostatics in the ATP-binding pocket. Global influences are those features remote to the binding pocket that nonetheless are capable of changing the shape of the ATP-binding pocket and hence the binding of inhibitors. There are approximately 20 amino acid side chain residues within 3–4 Å of kinase-bound ATP. Of these, six polar residues are invariant across all protein kinases and for many of the lipophilic residues only conservative replacements are seen [19]. Nonetheless, there is sufficient variation in these amino acid side chains, plus the additional residues that may be contacted by an inhibitor bound in the ATP site, to give rise to inhibitor selectivity. Relevant to the binding of many of the angiogenesis

inhibitors discussed in Table 1 is a ‘gatekeeper’ residue positioned adjacent to the residue in the linker region whose amide carbonyl binds to N-6 of adenine (in FGFR1 the linker hydrogen bonding residue is Glu562 and the gatekeeper is Val561). The 3,5-dimethoxyphenyl group of PD-173074 has been demonstrated by X-ray crystallography to bind into the pocket formed by a small gatekeeper residue (Val561) present in FGFR1 [7]. Specificity of inhibition is achieved because this aryl-binding pocket lies in a region of the ATP-binding pocket that is not used by ATP and because facile access to this aryl-binding pocket requires a small (Val, Thr or Ser) gatekeeper residue [20]. Although other ‘local’ ATP-binding site residues have been identified that effect inhibitor binding [21], the unique importance of the gatekeeper residue as a key local determinant for inhibitor selectivity for both tyrosine and serine/threonine kinases is widely recognized [18,22] and has been exploited by the Shokat group to

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Figure 2 Dendritigram of protein tyrosine kinase families and gatekeeper residues. The cytoplasmic tyrosine kinases are grouped as indicated in Table 1. Numbers indicate receptor tyrosine kinase class. Letters indicate identity of gatekeeper residue: green = small, red = large.

T, V

Cytoplasmic tyrosine kinases

M

11

2

M

M 1

T 13 14

T 16

10

T,V

8

T 9 T 12 7

15 I

M

5

F

V

4

L L

L 17 L

F

Letters indicate identity of gatekeeper residue Green = small Red = large

T

19

M

T

M

6, 18

M

Numbers indicate receptor tyrosine kinase class

3

V T

V Current Opinion in Chemical Biology

develop a novel chemical genetics approach for studying kinase signalling [23].

the EGFR. Similarly, PD-166285 illustrates a case in which a favourably small gatekeeper opens up angiogenic kinase families to inhibition by a compound that potently inhibits Src, a cytoplasmic tyrosine kinase [25]. Gleevec, a BCRABL kinase inhibitor and the first kinase oncology drug to be marketed, further exemplifies how a small (threonine) gatekeeper residue present in both the ABL and PDGF families can allow for cross-sensitivity of kinases with somewhat distant phylogenic homology [26,27]. In this example, the hydrogen-bonding properties of the hydroxyl side chain of theronine appear to be the key local determinant. A more clear-cut prediction can be made for the insulin receptor kinase family (class 2), which is both distil to classes 3–5 and has a larger methionine gatekeeper. In this case, none of the compounds in Table 1 show any appreciable inhibition of class 2 kinases. The combination of large gatekeeper residues and reduced homology appears to offer an adequate explanation for the insensitivity of the serine/threonine kinases to these inhibitors.

In addition to defining the protein kinase fold common to all kinases, the primary sequence encodes for several types of binding domains and sites of phosphorylation that regulate kinase structure and function. To the extent that primary sequence homology reflects this encoding of tertiary structure, family sequence homology might be expected to be a ‘global’ determinant of inhibitor selectivity. A phylogenetic tree organized to classify tyrosine kinases based upon the alignment of kinase domains is presented in Figure 2 (provided by Cell Signaling Technology on URL: http://www.cellsignal.com/retail/). This phylogenetic analysis makes readily apparent the relatedness of angiogenic kinase classes (3, 4, 5 and 10) [24]. The identity of the gatekeeper residue for both RTKs and cytoplasmic tyrosine kinases is also indicated on Figure 2. As suggested by this figure, the co-occurrence of a small gatekeeper residue and close family homology for classes 3, 4 and 5 is proposed to explain the pattern of inhibitor sensitivity seen in Table 1.

Efficacy of angiogenic tyrosine kinase inhibitors

The application of these two features — gatekeeper identity and phylogenic relatedness — while not an absolute predictor of kinase sensitivity, does offer useful insights. For example, the EGFR family (class 1) is more distal in its relationship to class 5, but retains a small gatekeeper residue. Although there is often little cross-reactivity between these two families, ZD-6474, which is a potent inhibitor of kinase VEGFR2, is also capable of inhibiting

There are now many examples of small-molecule RTK inhibitors currently in clinical trials that are proposed to work through an anti-angiogenesis mechanism. As described in the previous sections, none of these compounds are likely to be completely selective for their putative enzyme target. SU-5416 and SU-6668 are both VEGFR2 inhibitors, although SU-6668 is a relatively weak VEGFR2 inhibitor (PDGFR >> VEGFR2, see Table 1). SU-6668 also inhibits c-kit, FGFR and PDGFR activities

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in vitro and in vivo [11••,28–30]. The multiple RTK inhibitor, SU-6668, produced greater levels of apoptosis in tumour cells and tumour-associated endothelial cells than did the selective KDR inhibitor SU-5416 in a colon cancer liver metastasis model. In vivo studies with SU-6668 resulted in inhibition of receptor phosphorylation, growth rate for a large panel of human tumour xenografts, and regression of established tumours in nude mice. Tumours from animals treated with SU-6668 were severely depleted of microvessels and exhibited extensive apoptosis in tumour vessels shortly after administering treatment, within 6–24 h [11••,31•]. The VEGFR2 inhibitor listed in Table 1, ZD-6474, is 10-fold selective over EGFR and ~30-fold selective over PDGFRβ, and is also a very potent inhibitor of established tumour xenografts in nude mice, showing a clear dosedependant inhibition of tumour growth up to 79% at 100 mpk [10]. Tumour angiogenesis, primary tumour growth and spontaneous lung metastasis were all inhibited by ZD-6474 in an orthotopic murine renal cell carcinoma model [32]. Studies reported by Ciardiello et al. [9] indicate that inhibition of autocrine growth stimulation through TGFα/EGFR signaling contributes to tumour growth inhibition by ZD-6474 in some xenograft model systems where that pathway plays an important growth-promoting role. At the opposite end of the kinase selectivity spectrum lies CHIR200131, a very potent pan-RTK inhibitor [33,34]. This compound inhibits all three VEGF receptors and the FGF receptor with IC50 values below 10 nM and also inhibits the EGFR, PDGFRβ and c-kit with IC50 values in the 50–500 nM range. This compound exhibited very potent and broad-spectrum cell growth inhibition activity in standard cell growth proliferation assays. Proliferation of cell lines derived from human breast, lung, colon, ovary, prostate and brain carcinomas was inhibited at or below low micromolar concentrations of CHIR200131 in all cases. Antiangiogenic activity was observed in endothelial migration, tube formation and capillary vessel sprouting from rat aortic ring in vitro models, as well as matrigel in vivo models. Significant inhibition of tumour growth and metastasis and even regression were demonstrated in several xenograft model systems and combination treatments with 5-FU or CPT-11 were shown to be more efficacious than any single treatment alone. It’s not immediately clear why the cytotoxic agents provide added benefit in combination with CHIR200131 because CHIR200131 is already a potent cytotoxic agent for the cell lines utilized in the in vivo model systems. As indicated in Table 1, several of these broad specificity angiogenic kinase inhibitors have demonstrated sufficient preclinical safety to progress into clinical trials. This alone is not an unequivocal indication of the limited toxicity hoped for with anti-angiogenic therapy, as the life-threatening nature of cancer makes for lower barriers for clinical entry. However, the fact that no serious adverse effects were noted for any of the animal model systems described

for these compounds is a hopeful sign that a multiplex RTK inhibitor approach with limited toxicity may be possible.

Conclusion A common goal of modern drug discovery is to design a small-molecule inhibitor that can selectively target a single molecular process in a cell. Whether this target is an enzyme, receptor, protein–protein interaction, or otherwise, specificity for inhibition has been considered to be a key element of designing molecules that will be successful in the clinic. In reality, many commercially successful drugs interrupt multiple molecular processes. We suggest that the opportunity to design a multiplex inhibitor for kinases, which takes advantage of the both local and global features that shape the conserved ATP-binding site, is especially good. This targeting of the unique shapes of ATP-binding sites should enable a specific, yet multiplex, approach. The kinase inhibitor Gleevec is a multiplex inhibitor (ABL, KIT and PDGF); however, it is primarily used in a setting (chronic myeloid leukemia) where only one of the targeted kinases plays a critical role. The limitations of this approach are already apparent as evidenced by the appearance of single point mutations in the ABL kinase gene that confer drug resistance [35••,36•]. A multiplex inhibitor strategy, which targets multiple kinases that signal simultaneously in the same environment, should be more effective as it should be more difficult for tumour tissue to overcome the effects of the drug. It has been argued that inhibitors which target the angiogenic processes of the genetically stable endothelium should be less prone to develop resistance than those approaches that target the tumour. However, because multiple signaling pathways are activated by tumours to stimulate the endothelium, the application of a multiplex strategy may be required to achieve the optimal inhibition of tumour angiogenesis.

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