Protein kinase inhibition: natural and synthetic variations on a theme

219

Protein kinase inhibition: natural and synthetic variations on a theme Susan S Taylor* and Elzbieta Radzio-Andzelm

How a protein kinase is turned off is as critical for its

physiological function as is its catalytic activity. Examination of

solved crystal structures representing different protein kinase

subfamilies reveals a variety of strategies that are utilized by

nature to lock protein kinases into inactive conformations.

Pseudosubstrate and adenine mimetic mechanisms as well

as complementarity to surfaces other than the active site

are effective. Although most synthetic or natural product

inhibitors target the active site, specifically the ATP binding

site, a remarkably high degree of specificity can be achieved

which is due to the extended surface of the protein that these

inhibitors occupy. Although targeting of the ATP binding site

is proving to be very successful, there is also wide latitude for

designing inhibitors that target other surfaces of the kinases.

Addresses Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0654, USA *e-mail: [email protected]

Current Opinion in Chemical Biology 1997, 1:219-226

http://biomednet.com/elecref/1367593100100219

0 Current Biology Ltd ISSN 1367-5931

Abbreviations

C catalytic cdk cyclin-dependent kinase MAP mitogen-activated protein MLCK myosin light chain kinase PKA CAMP-dependent protein kinase PKG cGMP-dependent protein kinase PKI protein kinase inhibitor PRS2 peripheral recognition site 2 R regulatory

Introduction Fischer and Krebs [l] were the first to demonstrate, in

1955, that the reversible equilibrium between active and

inactive conformations of a protein could be regulated

by the post-translational addition of a phosphate moiety.

Not only are there thousands of proteins that utilize

this regulatory mechanism, but the number of protein

kinases that catalyze these phosphoryl transfer reactions

also number in the hundreds (reviewed in [Z]). It is

predicted that the human genome alone encodes over

2000 protein kinases. Since these enzymes regulate

almost every process in the eukaryotic cell by serving

as on/off switches, it is critical that every kinase be

tightly regulated physiologically. They are turned off and

on by very precise signals; thus if one could develop

strategies for selectively regulating a specific kinase by

therapeutic intervention, one could control an entire

signaling pathway. The mitogen-activated protein (MAP)

kinases, which are activated through complex but tightly

controlled kinase cascades in response to a number of

factors, including stress and cytokines, represent only one

set of targets where selective inhibitors are actively being

sought.

Although the catalytic core of protein kinases has been

evolutionarily conserved in all eukaryotic protein kinases

that phosphorylate serine, threonine, and tyrosine (re-

viewed in [Z]), the mechanisms by which the inhibition

of each kinase is achieved vary considerably. Although

the details differ, a continuing theme is the utilization

of modular domains for multiple functions. As the crystal

structures of more protein kinases have been solved, not

only of the catalytic cores but also of the cores plus

their regulatory domains, a variety of motifs for regulation

have been identified. These examples, summarized below,

not only provide a foundation for thinking more broadly

about the molecular features that regulate each kinase, but

also provide wide latitude for developing inhibitors which

extend beyond those obtainable using the conventional

approach of designing molecules that bind to the enzyme’s

active site. The purpose of this review is to summarize

some of the mechanisms used physiologically to achieve

protein kinase inhibition and compare them to the mech-

anism that is used by most known synthetic and natural

product inhibitors. Three specific inhibition mechanisms

will be discussed. Twitchin and CAMP-dependent protein

kinase (PKA) are examples of protein kinases that are

regulated by a pseudosubstrate mechanism; cdk2 and the

inactive insulin receptor provide examples of inhibition

that is achieved by an adenine mimetic mechanism; Hck

and Src provide examples of inhibition that is achieved by

locking the enzyme into an inactive conformation using

surfaces other than the active site. Inhibition mechanisms

are discussed in the context of the active enzyme and

the requirements for activity at the active site cleft. The

inhibitor domains can be part of the same polypeptide

chain, as in twitchin and Hck, or part of a separate subunit,

as in PKA and cdk2. In many cases these inhibitory

domains serve dual functions by inhibiting the kinase in

its inactive state and binding to other ligands in the active

conformation.

Structural features of the protein kinase core All known eukaryotic protein kinases share a conserved

catalytic core, in spite of considerable diversity in size,

mechanism of regulation and activation, and subcellular

localization. Since the structural features of this core in

its active conformation are best understood in functional

terms for PKA, one of the simplest protein kinases, we

shall use it as a prototype for the entire family. The

220 Next generation therapeutics

Modes of inhibition Pseudosubstrate inhibition

The mechanism of inhibition whereby the active site

of the kinase is occupied by an inhibitor peptide that

resembles a substrate was first suggested as the mechanism

for inhibition of the catalytic subunit of PKA by its

regulatory subunits (discussed in [14]). This strategy as

a general inhibition mechanism was more fully explored

by Kemp and co-workers [15] with myosin light chain

kinase (hILCK). The crystal structures of PKA [4] and

twitchin [16**] support this mechanism of inhibition. The

crystal structure of the C subunit of PKA w;1s first solved

as a binary complex with a peptide derived from the heat

stable protein kinase inhibitor (PKI) (residues 5-24). The

PKI sequence resembles the consensus sequence for PKA

substrates. As predicted by the binding of peptide analogs

to PKA [17], this pseudosubstrate occupies the active site

cleft [18]. In addition to this consensus sequence, which

alone is not sufficient to convey high affinity binding, PKI

has an essential amphipathic helix that lies amino-terminal

to the consensus site. This helix binds to a site peripheral

to the active site cleft. This peptide, PKI (residues 5-2-l),

binds with high affinity (Ki =0.2 nhl) in the presence of

ATP [17,19].

The regulatory (R) subunits of PKA contain a similar

consensus sequence, which is thought to occupy the

active site cleft in a similar manner. To achieve high

affinity binding, however, the R subunit interacts with

the surface on the C subunit that lies carboxy-terminal

to the consensus site. This region includes the essential

phosphorylation site, Thr197, and the basic residues that

surround this phosphate [ZO]. This site is designated as

peripheral recognition site 2 (PRSZ), to distinguish it from

the surface that is required by PKI [21]. Binding of these

inhibitors is thus bipartite. One segment that resembles

a substrate binds to the active site cleft. while a second

segment. contiguous in PKI but not contiguous in R, binds

to an additional site peripheral to the active site cleft.

Both PKI and R are competitive inhibitors with respect

to peptide substrates.

The structure of twitchin (Figure 2) provides probably

the most dramatic example to date of pseudosubstrate

inhibition [ 16”]. In this case, the conserved kinase core is

followed by an extended pseudosubstrate sequence, which

occupies the active site cleft as was predicted earlier. This

is followed by an IgG domain. The structure of the kinase

domain and the autoinhibitor segment alone define the

core and confirmed the pseudosubstrate mechanism of

kinase inhibition [ZZ], while the subsequent structure of

the kinase core plus the IgG domain shows the active

site completely occluded by the adjacent domain [ 16”].

The pseudosubstrate is docked into the active site cleft

while the IgG domain securely anchors itself on the

Protein kinase inhibition: natural and synthetic variations on a theme Taylor and Radzio-Andzelm 221

Figure 1

(b)

.oop

c 1997 Current Opmvx I” Chemical Biology

h Active conformation of the conserved core of the catalytic subunit of PKA. (a) In the center is the conserved core in its closed conformation wit

the two lobes (residues 40-l 27 and 126-300) shaded differently. The conserved residues (G50, G52, G55, K72, E91, Dl66, N171, D164,

E206, D220 and R260, in single letter code for amino acids) are shown as balls. ATP is bound at the base of the active site cleft between the

two lobes while the inhibitor peptide PKI (residues 5-241, shown in white, binds on the surface of the large lobe. (b) An expanded view of the

small lobe where the central role of the glycine-rich loop is emphasized. The glycine-rich loop (residues 49-57) and the linker that joins the two

lobes (residues 120-l 27) are shown in black. The structures were generated using PDB file 1 ATP.

surface that lies carboxy-terminal to the active site cleft.

In hlLCK. a related family member, the pseudosubstrate

peptide domain overlaps with a calmodulin binding site

(discussed in [23]). When Car+ is bound to calmodulin,

the CaZ+/calmodulin complex competes favorably with the

inhibitory domain, thus exposing the active site of the

enzyme. Twitchin is activated in a similar fashion, by

binding to another protein complex, CaZ+/SlOO [El].

PKA and twitchin are assembled as fully active modules

that are maintained in an inactive state by formation of

a stable complex with an inhibitor. In the case of PKA,

the inhibitor is a separate protein, whereas with twitchin

(and IZILCK) the inhibitor is a contiguous domain of the

kinase itself. In both cases, a secondary messenger causes

the release of the inhibitor segment [14,23].

Adenine mimetic inhibition

While pseudosubstrate inhibition is very attractive and

logical, it is not the only mechanism by which kinase

inhibition is achieved. Another mechanism that has onlv

been recognized more recently utilizes a peptide segment

that mimics the adenine ring of ATP. This pepcide

functions as an adenine mimetic and fills the shielded

pocket that is typically occupied by the nucleotide

base in the active kinase. To date, the best example

of this mechanism is represented by the structure of

p27Kllll bound to the cdk2-cyclin complex [ZS”]. In this

structure p27KiPl spans the surface of both cdk2 and

cyclin (see Figure 3). The carboxyl terminus of p27Q~l

specifically folds over onto the small lobe of the kinase

core. The glycine-rich loop, a key component of the

nucleotide positioning motif, becomes a distorted and

discontinuous part of the p sheet. The Tyr88 residue

of p27Kipl folds into the adenine binding pocket and

hydrogen bonds with the backbone carbonyl of Glu81

and the backbone amide of Leu83, both in the short

linker strand that joins the two lobes. A comparison

of the active site filled by ATP and the corresponding

adenine binding pocket of cdkZ-cyclin-p27Kil’l filled with

the Tyr88 of p27Kipl is also shown in Figure 3. How

widely this mechanism is utilized by other protein kinases

must await crystal structures of other inhibited enzymes.

However, given the critical importance of protein-protein

interactions for kinase signaling pathways. this is likely to

be used repeatedly.

The structure of a truncated form of the insulin receptor

provides another example in which a peptide sequence can

fold into the active site cleft and mimic the adenine ring of

ATP, thus inhibiting enzymatic activity [26]. In this case,

it is Tyrl158, part of the activation loop, that folds into

the adenine binding pocket. The tyrosine hydroxyl makes

contacts similar to those seen in the cdk2-cyclin-p27W~~

complex. This crystal structure suggests an intriguing

mechanism whereby the dephosphorylated tyrosines in

the activation loop stabilize the inactive conformation

of the enzyme. The full physiological relevance of this


Figure 2

Autoregulation of the twitchin and Hck kinases [16”,26”]. Autoregulation (inhibition of kinase activity) is achieved using two different

mechanisms that result in the active site being maintained in an inhibited state by domains that flank the core. (a) The structure of the twitchin

kinase core joined to the autoinhibitor segment and the IgG domain. The autoinhibitor segment (residues 6199-6255) is locked firmly into

place at the active site cleft by numerous contacts and by the positioning of the IgG domain. (b) The structure of the Hck kinase core with its

amino-terminal &c-homology flanking SH2 and SH3 domains. In this case the SH2 and SH3 domains bind to Tyr526 in the carboxy-terminal

tail and to the linker strand that joins the SH2 domain to the core, respectively. In this structure of the inhibited kinase, the active site cleft is

fully exposed but locked into a conformation that cannot support catalysis. The structure of twitchin was generated using PDB file 1 KOA. The

coordinates for Hck were kindly provided by John Kuriyan (The Rockefeller University, New York).

mechanism for the insulin receptor awaits a structure

solution of the full-length protein, since both membrane

localization and the carboxy-terminal tail may contribute to

the conformation of the core. The physiological relevance

of this mode of inhibition is also unclear since in the cell,

where ATP concentrations are high, ATP may displace

Tyr1158 and lead to structural instability. It is clear,

however, that the adenine binding pocket can be an

important potential target for physiological inhibition.

Other mechanisms for locking the kinase core in an

inactive conformation

The recently solved structures of two nonreceptor tyrosine

kinases, Src and Hck, illustrate another novel mechanism

for locking the catalytic core into an inactive conforma-

tion [27”,28**]. These structures (of the core plus the

contiguous Src-homology domains, SH2 and SH3,) also

emphasize the importance of having a full length protein

structure as opposed to structures of isolated domains. It

is very easy to overinterpret the significance of crystal

packing in isolated domains when the natural partner, in

this case the kinase core, is missing. In the case of Src

and Hck, the finely tuned inhibition mechanism was only

revealed when the structures of the proteins containing all

three domains were solved [27**,28”].

The orientation of the Hck kinase core relative to the

SH2 and SH3 domains is shown in Figure 2b. This

conformation is stabilized by two specific interactions:

the phosphorylated Tyr at the carboxy-terminus, Tyr527,

is hydrogen bound to the adjacent SH2 domain, and

the proline-rich segment (which links the SH2 domain

to the kinase core) serves as an intramolecular docking

site for the SH3 domain. Consequently, the kinase core

is physically constrained in a conformation that cannot

support catalysis. In addition to restricting the conforma-

tional flexibility of the core, two specific regions of the

kinase core are either incorrectly aligned or disordered.

The activation loop is disordered because it is not

phosphorylated [B”]. Activation of Src correlates directly

with the phosphorylation of Tyr416 in its activation loop

[29]. In addition, the C helix in the small lobe is twisted so

that the conserved Glu310 is facing away from Lys295, its

partner in the active enzyme. Instead Glu310 is exposed

to the solvent and faces the activation loop, where it

actually forms an ion pair with the highly conserved

Arg385, which immediately precedes the catalytic loop.

Following phosphorylation of Tyr416, this same arginine

helps to assemble the activation loop into its correct

and active form (in the inhibited conformation the same

residue helps to stabilize the inactive enzyme). Correct

phosphorylation of this loop is essential for full activity of

most protein kinases (reviewed in [30’]). In PKA, lack of

this essential phosphate (on Thr197) results in a decrease

in the efficiency of phosphoryl transfer and an increase

in the Km for ATP [31]. In spite of its distance from the

active site cleft, this phosphate is linked by an extensive

network of interactions to most of the residues involved in

recognition of both ATP and peptide substrates, as well as

those involved in catalysis.


Figure 3

fb) Beta 3

Linker

p27(84-93)

Linker ’ ATP

Structural views of the cdk2 kinase bound to an inhibitor, p2i’W [25”1. (a) This structure shows cdk2-cyclin A bound to an inhibitor peptide,

p2Wpt, and reveals a novel mechanism of inhibition. p27W flanks the surface of both cdk2 and cyclin A with the p27W carboxy-terminal tail

binding to the adenine binding pocket of cdk2. (b) A view of the adenine pocket with Tyr88 hydrogen bonding to the linker strand and Lys33 of

the kinase core. In this conformation the glycine loop is very distorted. (c) The corresponding view of this region when ATP is bound, rather than

p27W. to the cdk2-Cyclin A complex. Amino acid residues are designated using single letter code in the figure. The coordinates for cdk were

kindly provided by Nikola Paveltich (Memorial Sloan-Kettering Cancer Center, New York).

The role of the SH3 domain in maintaining the kinase

core in an inactive conformation was demonstrated clearly

by a kinetic analysis of the effect of Nef [32], a small,

(25-27 kDa) protein required for infection by HIV. It

binds with high affinity to the SH3 domain of Hck. Nef

is a potent activator of the Src kinase by a mechanism

that specifically involves displacement of the SH3 domain

from the kinase core. This represents a new strategy for

regulation of Src activity-a strategy that targets a region

that is quite distinct from the active site.

Synthetic and natural inhibitors A number of synthetic and natural product kinase

inhibitors have now been identified, and so far all compete

with ATP for binding. Thus, like p27Kip1, they have

a subsite that mimics the binding of adenine. Despite

being competitive inhibitors with respect to ATP, however,

and in contrast to earlier predictions, a number of

these inhibitors show exquisite specificity. Particularly

impressive examples are the pyridinylimidazole inhibitors

that specifically target ~38, a MAP kinase [33]. Other

examples are the pyrrolo- and pyrazoloquinazolines that

are specific for the epidermal growth factor (EGF) receptor

(reviewed in [34]; [35]). Thus, in spite of a highly

conserved core, a few critical differences allow for high

specificity. Because the nucleotide is deeply embedded in

the active site cleft and extends over such a large surface

area, there are potentially many ways to achieve specificity.

As seen in Figure 1, the entire nucleotide-including the

adenine, ribose, and triphosphate moieties -is specifically

embraced by the glycine-rich loop. Crystal structures of

several of these synthetic inhibitors have now been solved,

and these structures reveal at least one common feature.

All of these inhibitors target the adenine binding pocket

whereby one of the rings mimics the planar adenine ring,

fills the pocket, and also forms hydrogen bonds to the

linker strand that joins the small and large lobes. This

hydrogen bonding is to the same backbone carbonyl and

amide that are complemented by the N6 and Nl nitrogens

of the adenine ring of ATP.

A comparison of the binding of two such inhibitors is

shown in Figure 4. H7, H8, and H89 are isoquinoline

sulfonamide inhibitors of PKA, PKI, and PKG. H89 shows

the highest specificity for PKA (Ki =O.SnM) [36]. By

solving the crystal structures of each of these inhibitors

in complex with the C subunit of PKA, Engh et a/. [37-l

showed that the isoquinoline ring mimics the adenine ring

of ATP in the ternary complex of CATP.PKI (5-24). The

nitrogen in the isoquinoline ring hydrogen bonds to the


linker strand, mimicking the hydrogen bond made by the

Nl nitrogen of the adenine ring of ATI? The higher degree

of selectivity in H89, compared to H7 and H8, is due to

an extended region, including a bromine, that interacts the

glycine-rich loop, resulting in distortion of the loop.

The crystal structures of cdk2 bound to three different

inhibitors also reveal a common mechanism of inhibition

[38,39]. All have rings that occupy the adenine binding

pocket, and most also form hydrogen bonds with the

linker strand. The orientation of the rings differs, however.

Olomoucin, a highly specific inhibitor, actually contains

a purine ring like ATP, but differs because there is a

bulky substitution on the N6 nitrogen. As a consequence

of this bulky substituent, the olomoucine purine ring is

oriented in a configuration that is reversed compared to

the purine ring of ATP [39]. In this case, the hydrogen

bonding involves the N3 and N9 nitrogens in the purine

ring which replace the N6 and Nl nitrogens in ATI?

The indolines are another group of highly specific

ATP-based inhibitors. Some of these compounds contain

an oxindole core, and are specific for the fibroblast growth

factor (FGF) receptor tyrosine kinase [40*]. The structure

of this complex reveals that the oxindole ring binds to the

adenine pocket of the FGF receptor and hydrogen bonds

to the backbone of the linker strand.

Specific inhibition of the ~38 kinase by pyridinylimida-

zoles is one of the best examples where the molecular

Figure 4

basis for the specificity of this inhibition is understood.

These compounds were first identified by screening for

inhibitors of the lipopolysaccharide-induced immune re-

sponse. In a classic search for the target of these inhibitors,

Lee et al. [33] identified ~38, a member of the MAP

kinase family. The selectivity of these inhibitors has been

well documented [41]; however the actual basis for the

selectivity remained unclear until crystal structures were

solved. The crystal structure of ~38 is similar to that of

ERK-2, another MAP kinase [42,43]. It was the structure

of ~38 bound to VK-1911, a pyridinylimidazole, however,

that clearly correlated specificity with a single amino

acid difference in the ATP-binding pocket [44*-l. The

structure-based prediction was confirmed by mutagenesis

of this amino acid [44**].

Conclusions Protein kinases are clearly important targets for drug

design. Earlier predictions that achieving specificity would

be difficult given the highly conserved nature of the kinase

core are clearly not valid. So far most synthetic inhibitors

target the adenine binding pocket at the active site cleft

and compete with ATP for binding. Despite this, high

specificity can be achieved by this strategy [33,34,36,40*].

As the structures of more inhibitor-kinase complexes are

solved, rules will hopefully be defined which will allow

rational design of inhibitors that will target a specific

protein kinase. We are rapidly moving in that direction.

The conserved nature of the kinase core also makes it an

excellent candidate for homology modeling, whereby the

(a) (b)

Structure of synthetic inhibitors bound to various protein kinases reveal a common mechanism for binding to the adenosine binding pocket at

the active site cleft [37-l. (a) Shows ATP binding to the C subunit of PKA superimposed with H8 binding to the same pocket. H8 is shown as a

thick line. and ATP is shown as a thin line. This structure was generated using PDB file 1YDS. (b) Shows H89 binding to the active site clefi of

the C subunit of PKA superimposed with ATP bound to the same pocket. In this case, H89 is shown as a thin line and ATP is shown as a thtck

line. This structure was generated using PDB file 1 YDT.


sequence of the conserved core of any protein kinase can

be modeled onto a three-dimensional structural template

[15]. Once again, as more structures are solved, the

accuracy of the modeling also will improve. It is also

clear, however, that the design of specific inhibitors need

not target only the active site cleft. The C helix, the

activation loop or the segments that flank the kinase core

are equally valid targets for drug design, as is targeting the

ligand binding sites at the active site. Although designing

inhibitors that bind to the active kinase is one strategy,

designing or screening compound libraries for inhibitors

that prevent kinase activation is an equally plausible

strategy - one that has been explored by nature but yet

to be exploited by combinatorial chemistry.

Acknowledgements SW is supported by grants from the National Institutes of Health and the American Cancer Society. ER-A was supported in part by National Institutes of Health ‘liaining Grant I’HS ‘1‘32 DK07233. The authors wsh to thank N Narayana and T Diller for helpful comments and discussions.

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This set of structures shows the H-series inhibitors bound to the catalytic subunit of CAMP-dependent protein kinase.

36.

39.

40. .

Filgueira de Azevedo W Jr, Mueller-Dieckmann H-J, Schulze- Gahmen U, Worland PJ, Sausville E, Kim S-H: Structural basis for soecificitv and ootencv of a flavonoid inhibitor of human CDKi a cell-cycle ‘kinase: Proc Nat/ Acad Sci USA 1996, 93:2735-2740.

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An example of specific kinase inhibitor that docks to the active site of the fibroblast growth factor receptor.

41. Cuenda A, Cohen P, Buee-Scherrer V, Goedert M: Activation of stress-activated protein kinase 3 (SAPKS) by cytokines and cellular stresses is mediated via SAPKKS (MKKG); comparison of the specificities of SAPKB and SAPK2 (RK/p38). EMBO J 1997, 16:295-305.

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44. Wilson KP, McCaffrey PG, Hsiao K, Pazhanisamy S, Galullo V, . . Bemis GW, Fitzgibbon MJ, Caron PR, Murcko MA, Su MSS:

Structural basis for specificity of pyridinylimidazole inhibitors of p38 MAP Kinase. Chem Biochem 1997, 4:423-431.

This structure of a p36-specific inhibitor bound to ~36 provides an excellent example of specificity. Mutagenesis of a single residue functionally confirms the structural basis for specificity.

Protein kinase inhibition: natural and synthetic variations on a theme

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