Selective 3-Phosphoinositide-Dependent Kinase 1 ... - ACS Publications

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Selective 3‑Phosphoinositide-Dependent Kinase 1 (PDK1) Inhibitors: Dissecting the Function and Pharmacology of PDK1 Miniperspective Jesús R. Medina* Oncology Research, GlaxoSmithKline, Collegeville, Pennsylvania 19426, United States ABSTRACT: 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a protein target that has generated considerable interest in both academia and the pharmaceutical industry. PDK1 is responsible for regulating the activity of related kinases in the AGC kinase family, including AKT, by phosphorylating a specific threonine or serine residue within the activation loop which is critical for kinase activation. Many of the kinases activated by PDK1 regulate cellular process such as cell survival, differentiation, growth, and protein expression. Although significant work has been done to understand the role of PDK1 function in cells, recently discovered potent and selective small molecule PDK1 inhibitors are providing a unique opportunity to further dissect PDK1 function and predict the pharmacological consequences of PDK1 inhibition. This Miniperspective reviews the discovery of these selective PDK1 inhibitors and highlights their value in cellular studies, the understanding of PDK1 biology, and the impact on the therapeutic potential of PDK1 inhibition in cancer.

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INTRODUCTION Target validation is a critical step in the drug discovery process.1 Over 50% of failures at phase II and phase III clinical trials across all therapeutic areas are attributable to a lack of efficacy that could be a result of the target being nonoptimal for therapeutic benefit.2 Therefore, the prediction of which protein targets will be disease modulating through comprehensively performed target validation studies should help to reduce attrition rates during drug development. In oncology, there is a tremendous need to identify novel and significantly improved drugs, and understanding the signaling pathways involved in cancer cell proliferation and survival is critical to finding predictive factors for tumor aggressiveness, prognosis, and treatment.3 Gene silencing and expression of functionally impaired mutant forms of specific proteins have frequently been used to assess protein function in cells.4 However, the availability of cell-permeable, potent, and highly selective inhibitors for a potential drug target is highly desired to assess its tractability, as cellular phenotypes observed by genesilencing technologies are not necessarily reproduced by pharmacological inhibition of a particular target.5,6 For instance, gene silencing alters the expression of their target protein, which may disrupt protein complexes or impair protein functional domains that may be unaffected by small molecule inhibition. In addition, since the target has been ablated for days or at the start of embryonic development, the cellular phenotypes observed may be indirect and result from long-term perturbations of gene expression.7 Unfortunately, the discovery © XXXX American Chemical Society

of highly selective small molecule inhibitors is very challenging, and most small molecule inhibitors lack the strict selectivity necessary for high confidence in the pharmacological response. Therefore, the use of these nonspecific compounds to address protein function could lead to misinterpretation of data, as the pharmacological phenotype may just reflect “off-target” effects of the molecule.8 Kinases have become one of the most intensively pursued classes of drug targets. This has generated considerable interest in the development of small molecule kinase inhibitors to be used for the treatment of cancer and other diseases and as probes to understand the function of these enzymes.9 However, the design of potent inhibitors with high selectivity is particularly challenging in the kinase field, since approximately 518 protein kinases are encoded in the human genome, all sharing similar structural frameworks in the ATP-binding site, with which most small molecule inhibitors interact.10,11 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a kinase target that has generated considerable interest in both academia and the pharmaceutical industry.12−14 PDK1 is the protein kinase responsible for regulating the activity of related kinases in the AGC [protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC)] kinase family, including AKT, by phosphorylating a specific threonine or serine residue within the activation loop (T-loop) which is critical for kinase Received: January 4, 2013

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dx.doi.org/10.1021/jm4000227 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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activation.15 Many of the kinases activated by PDK1 regulate cellular processes such as cell survival, differentiation, growth, and protein expression, in response to second messenger signals. PDK1 was discovered as a phosphoinositide 3-kinase (PI3K) dependent enzyme that had the ability to phosphorylate AKT.16 This process was determined to be dependent in vitro on the concentration of phosphatidylinositol 3,4,5-trisphosphate (PIP3), the resulting product of the PI3K catalyzed phosphorylation of phosphatidylinositol 4,5-bisphosphate at the 3′ position.17 Activation of PI3K by growth factor signaling results in the production of phosphatidylinositol 3,4-bisphosphate and PIP3, which colocalize AKT and PDK1 to the plasma membrane through interaction with their respective pleckstrin homology (PH) domains, thus allowing PDK1 to phosphorylate AKT at the T-loop residue Thr-308 in a PIP3dependent manner (Figure 1).15 Binding of PIP3 to AKT also

growth was observed in hypermorphic PDK1 mice, adding additional evidence supporting the potential therapeutic benefit of PDK1 inhibition in cancer therapy.21 Compounds 1 (OSU-03012) and 2 (OSU-03013) (Figure 2) are two low micromolar PDK1 inhibitors published in 2004 that were designed by structure-based optimization of celecoxib.22 However, the first report on the biological characterization of potent submicromolar small molecule inhibitors of PDK1 (e.g., 3 (BX-320) and 4 (BX-795)) was reported by researchers at Berlex Biosciences.23 The kinase selectivity for these compounds was determined by their effect on the in vitro activity of 10 different Ser/Thr and tyrosine kinases, including the related AGC kinases PKA and PKCα. Although both compounds 3 and 4 were initially described as specific inhibitors of PDK1 (Figure 2), displaying greater than 20-fold selectivity for PDK1 relative to the kinases in this panel, subsequent profiling studies by other groups revealed these compounds as potent inhibitors of other kinases. For instance, in a 72 protein kinase profiling study, compound 4 (PDK1 IC50 = 17 nM in this study) exhibited more potent inhibition toward Aurora B (IC50 = 11 nM), IKKε (IC50 = 9.5 nM), and TBK1 (IC50 = 2.3 nM), creating a shadow of uncertainty on any conclusions made about the role of PDK1 in cell based studies using these compounds.10 For example, compound 4 was shown to cause a cell cycle arrest in G2/M and to inhibit tumor formation. However, studies by researchers at UCSF Cancer Research Institute revealed that the ability of 4 to cause G2/M arrest was similar in PDK1+/+ embryonic stem (ES) cells compared to PDK1−/− ES cells, suggesting that the cell cycle consequences observed when using this compound were not associated with PDK1 inhibition.24 Similar to the previous profiling studies of 4 against 72 protein kinases, profiling against a larger panel of 211 protein kinases revealed that several kinases known to influence cell cycle control such as CDK1, CDK2, and Aurora A/B/C were strongly inhibited by 1 μM 4, suggesting at least one of these kinases as the relevant target responsible for G2/M arrest and not PDK1.24 In 2008, a comprehensive review article on small molecule inhibitors reported to interact with PDK1 was published by Peifer and Alessi.14 Although various structural classes of small molecule PDK1 inhibitors were published by that time (Figure 2), none of them seemed to have high selectivity for PDK1. Since then, a variety of other PDK1 inhibitors have been reported,25−28 with only a few showing high selectivity for PDK1 (Figure 3).29−32 In this article, we will review the discovery of these highly specific small molecule PDK1 inhibitors, highlighting their value in cellular studies, the understanding of PDK1 biology, and the impact on the therapeutic potential of PDK1 inhibition in cancer.

Figure 1. PDK1 mediated signal transduction.

induces conformational changes that facilitate PDK1 phosphorylation.18 However, many other kinases now known to be PDK1 substrates are phosphorylated and hence activated by PDK1 in a PIP3-independent manner. In contrast to AKT, these kinases lack a PH domain and are therefore not dependent on colocalization with PDK1 at the plasma membrane. For these kinases, phosphorylation at the hydrophobic motif (HM) by distinct upstream kinases enables PDK1 to recognize and interact with these enzymes through its PDK1 interacting fragment (PIF) pocket, a small phosphate binding groove located in the PDK1 catalytic domain. This interaction facilitates the T-loop phosphorylation of these substrates (Figure 1).19 Since a large fraction of the AGC family appears to be regulated through phosphorylation by PDK1, it has been named the “master regulator” of the AGC signal transduction.20 PDK1 phosphorylates several kinases associated with PI3Kand mitogen-activated protein kinase (MAPK) signaling pathways involved in cell survival and proliferation. Therefore, pharmacological inhibition of PDK1 was predicted to inhibit oncogenic cellular process and thus be therapeutically beneficial in cancer treatment.13,15 This hypothesis was supported by findings that most human tumors have mutations and/or loss of genes such as phosphatase and tensin homologue deleted on chromosome 10 (PTEN), resulting in hyperphosphorylation and activation of PDK1 substrates involved in proliferation and tumor growth.13,15 Moreover, a decreased incidence of tumor

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GLAXOSMITHKLINE INHIBITOR In 2011, we disclosed at GlaxoSmithKline the discovery of potent and highly selective PDK1 inhibitors.29 In particular, compound 8 (GSK2334470) exhibited excellent selectivity for PDK1. When tested against an in vitro kinase selectivity panel of 285 protein and lipid kinases, only 24 kinases were inhibited >50% in the presence of 10 μM 8. Moreover, compound 8 exhibited >1000-fold selectivity for PDK1 when compared against a subset of 13 kinases including members of the AGC family such as AKT1 (IC50 > 10 000 nM) and ROCK1 (IC50 = 7943 nM) and kinases that influence cell cycle such as Aurora A (IC50 = 39 810 nM) and Aurora B (IC50 = 3162 nM).29 B



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Figure 2. Selected PDK1 inhibitors disclosed in the literature by 2008.

The discovery of 8 started with a fragment-based lead discovery campaign (Figure 4).33 A focused library of 1065 fragments with hydrogen bond donor and/or acceptors motifs designed to engage the kinase hinge was screened using a biochemical kinase assay at high fragment concentration (400 μM). Compounds with IC50 < 400 μM, chemical purity of ≥98%, ligand efficiency LE > 0.40, and chemical tractability were selected for further evaluation by saturation transfer difference (STD) experiments to confirm binding to PDK1. Subsequent crystallography and focused mining of our compound collection led to the discovery of a highly ligand efficient aminopyrimidine-aminoindazole 14, a leadlike derivative of the aminoindazole fragment hit 13 (Figure 4). The retained high LE exhibited by 14 relative to 13 can be explained by the X-ray structure determination of 14 bound to PDK1 which revealed that every heteroatom/polar group in 14 is engaged in productive hydrogen bond interactions with the active form of PDK1.33 Examination of the X-ray crystal structure of 14 bound to PDK1 suggested that substitution at the 6-position of the pyrimidine ring could fill the lipophilic pocket under the

Figure 3. Potent and selective PDK1 inhibitors disclosed in the literature.

Figure 4. GlaxoSmithKline fragment-based lead discovery campaign. C



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Figure 5. Lead optimization effort toward compound 8.

the G-loop (Figure 6). Compound 17 also retained a high degree of kinase selectivity associated with compounds having disubstituted amines attached at the 6-position of the pyrimidine ring.29 Additional structural information suggested that favorable interactions with another lipophilic pocket along the G-loop could be accessed through carboxamide substitution at the βposition of either a morpholine or piperidine ring attached at the 6-position of the aminopyrimidine ring. This hypothesis proved to be correct as represented by compound 18 (Figure 5) and led us to the preparation of cis-disubstituted sixmembered ring analogues containing both the β-carboxamide and the α-methyl substituent. Further SAR revealed that methyl substitution on the pyrimidine 2-amino group provided compounds with increased cellular potency and kinase selectivity leading to the discovery of 8 (Figure 5).29 Alessi and co-workers at the University of Dundee, U.K., further studied 8 in cellular systems to assess its ability to block the phosphorylation of a range of PDK1 substrates.34 They confirmed the high specificity of 8 by studying its effect on the activity of 95 protein kinases, including 13 AGC kinases closely related to PDK1, and 15 lipid kinases. At 1 μM, no other kinase tested was significantly inhibited, which was 100-fold higher than the IC50 of inhibition of PDK1 in activating full-length AKT in this study (PDK1 IC50 ≈ 10 nM). In human embryonic kidney 293 (HEK-293) cells stimulated by insulin-like growth factor 1 (IGF1), which induces a strong activation of the PI3K pathway, 8 suppressed to basal levels within minutes the T-loop phosphorylation and activation of the cytosolic substrates serum- and glucocorticoid-induced protein kinase (SGK) and p70 ribosomal S6 kinase 1 (S6K1), which do not bind PIP3, at doses of 0.1 and 1 μM 8, respectively. In addition, 8 inhibited at similar doses the phosphorylation of N-myc downstreamregulated gene 1 (NDRG1) and S6 protein, physiological

glycine-rich loop (G-loop). We noticed that substitution with monosubstituted amines provided a high level of potency and LE, while disubstituted amines attached at the 6-position conferred a higher level of kinase selectivity. These observations, together with structural information from the Xray crystal structures of PDK1 bound monoalkyl (15) and dialkylamine (16) substituted analogues at the 6-position of the aminopyrimidine ring, led to the design of the α-methyl substituted morpholine derivative 17 (Figures 5 and 6). This compound exhibited the higher potency observed for monosubstituted amine analogues, most likely due to positioning of the α-methyl group into a small lipophilic pocket under

Figure 6. (A) Overlay of X-ray crystal structures of PDK1 bound to 15 (green) and 16 (blue). (B) X-ray crystal structure of 17 bound to PDK1. D



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Figure 7. Lead optimization effort toward compounds 9 and 10.

required 100- to 1000-fold lower levels of PDK1 relative to other PDK1 substrates not having a PH domain.35,36 However, recent studies by Alessi suggest that PIF-pocket dependent pathways are also operating to enable AKT activation in response to stimuli that activate the PI3K pathway.37 In this mechanism, the AKT HM is first phosphorylated at Ser-473 by mammalian target of rapamycin complex 2 (mTORC2). The phosphorylated HM of AKT is then able to dock into the PDK1 PIF pocket, allowing PDK1 to phosphorylate Thr-308 at the AKT T-loop in a similar way as all PDK1 cytosolic substrates are activated. Consistent with this hypothesis, mutation of the AKT HM residue Ser-473 to alanine resulted in almost complete inhibition of Thr-308 phosphorylation in IGF-1-stimulated HEK-293 cells at 0.3−1 μM doses of compound 8, while no significant inhibition was observed for wild-type AKT even at a 10 μM dose of 8.37 This ability of PDK1 to activate AKT via two alternative mechanisms [(1) PDK1 and AKT binding to PIP3; (2) PDK1 PIF-pocket interacting with AKT Ser-473 after it is phosphorylated by mTORC2] could result in the efficient activation of AKT by even residual amounts of noninhibited PDK1, making it very difficult to suppress AKT activation by PDK1 inhibitors such as 8 as single agents. Consistent with these observations, researchers at Merck demonstrated that RNA interference mediated 90% knockdown of PDK1 was not sufficient to block PI3K activation in various tissues and did not prevent tumor formation and progression resulting from PTEN loss.38

substrates of SGK1 and S6K1, respectively. Interestingly, although 8 also suppressed the T-loop phosphorylation and activity of the cytosolic substrate p90 ribosomal S6 kinase 2 (RSK2), the inhibition required extended incubation with the drug (8−24 h).34 This result could be explained by the potential differential kinetics of phosphatase mediated T-loop dephosphorylation of the various PDK1 substrates. Perhaps the most striking result from Alessi’s characterization of compound 8 was derived from the cellular studies of effects on AKT phosphorylation. Compound 8 blocked endogenous AKT1 Thr-308 phosphorylation in HEK-293 cells at ∼1 μM under conditions of low PI3K activity. However, 8 failed to inhibit the Thr-308 phosphorylation of endogenous or overexpressed full length AKT even at 3 μM in response to the PI3K pathway activator IGF1, which results in increased levels of PIP3. Consistent with these results, a dose of 3 μM 8 only marginally suppressed Thr-308 phosphorylation or AKT activation (∼3-fold) in U87 glioblastoma cells that lack PTEN expression and therefore should also have increased levels of PIP3. In these PTEN deficient cells, 1 μM 8 maintained the ability to efficiently inhibit the phosphorylation of the PIP3independent SGK1 substrate NDRG1, similar to the observations in HEK-293 cells.34 These results are particularly striking considering that a great amount of the interest in finding PDK1 inhibitors for the treatment of cancer was due to the belief that they could inhibit AKT, especially in response to stimuli or mutations that induced large activation of the PI3K pathway. The failure of compound 8 to inhibit AKT in response to conditions that stimulate the PI3K/AKT pathway was initially explained by the mechanism by which PDK1 activates AKT. Contrary to other AGC kinases (represented in Alessi’s study by SGK, S6K1, and RSK2), AKT phosphorylation by PDK1 is regulated directly by the interaction of both PDK1 and AKT PH domains with PIP3. This interaction colocalizes both PDK1 and AKT to the plasma membrane and therefore increases the probability of interaction between both enzymes, requiring only a fraction of endogenous PDK1 to activate AKT. Consistent with this hypothesis, previous biochemical studies demonstrated that activation of AKT by PDK1 in the presence of PIP3-containing lipid vesicles

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PFIZER INHIBITORS Another PDK1 inhibitor binding to the active form of the kinase was published by Pfizer in 2011.30 Inhibitor 19 was identified from a screen of the Pfizer corporate file (Figure 7). Guided by X-ray crystallography, the program concentrated on obtaining analogues selective against PI3Kα, since inhibition of this kinase could also reduce the amount of phosphorylated AKT at Thr-308 and confound the observed effects of PDK1 inhibition. Initial medicinal chemistry efforts identified compound 20 as a promising lead for building selective compounds (Figure 7). The X-ray cocrystal structure of 20 bound to PDK1 revealed the pyridyl group filling the pocket E



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terminal aryl ring with a fluorine to generate compound 22, which was hypothesized to be selective because of steric interference with Pro-810 of PI3Kα. The lipophilicity of 22 was lowered by replacing the methylenes in the cyclopentyl ring with heteroatoms. This effort led to compounds 23, 9, and 10, which exhibited a reduced clogP by more than 2 units while maintaining selectivity (Figure 7). Both analogues 9 and 10 showed a significant boost in selectivity. The in vitro cellular potency (reduction of phosphorylated AKT at Thr308) for these three compounds was determined in SKOV3 and A549 cell lines. Both fluorinated analogues 23 (SKOV3 IC50 = 70 nM; A549 IC50 = 50 nM) and 9 (SKOV3 IC50 = 70 nM; A549 IC50 = 80 nM) showed higher cellular potency than des-fluoro analogue 10 (SKOV3 IC50 = 200 nM; A549 IC50 = 300 nM). These three compounds were profiled against a panel of 36 kinases to assess their broader selectivity. Both fluorinated compounds inhibited five other kinases >70% at 1 μM, while the des-fluorinated analogue 10 only inhibited one other kinase (Chk2). The IC50 values were measured for four kinases (MARK1, Aurora A, TrkA, and GSK3β) that might contribute to antiproliferative effects in culture. All three analogues exhibited >100-fold selectivity against these four kinases. However, compound 23 exhibited a potency of ∼100 nM against Aurora A, MARK1, and PI3K which could potentially contribute to antiproliferative activity in cellular assays. The antiproliferative activity for the most selective acetamide analogues 9 and 10 (off-target kinase IC50 values of >340 nM) was measured in A549 and H460 cell lines. Similar to findings by GlaxoSmithKline with compound 8, these compounds showed only modest effects in preventing tumor cell growth.29,30,39

under the G-loop, analogous to the α-methyl substituent in the GlaxoSmithKline inhibitors (Figure 8).29,30 To improve

Figure 8. Overlay of X-ray crystal structures of PDK1 bound to 17 (blue) and 20 (green).

potency, the flexible ethyl linker, which needed to be in the energy unfavorable gauche conformation to position the aryl ring under the G-loop and in the bioactive conformation, was locked into a five-membered ring. In addition, the pyridine ring was replaced with a phenyl ring, since unblocked pyridines are associated with increased risk of CYP inhibition, and the phenyl analogue of 20 was equipotent. These efforts resulted in the ring-locked analogue 21, which exhibited a significant improvement in both potency and selectivity (Figure 7). Additional selectivity was achieved by substitution of the 4-position of the

Figure 9. Sunesis and Biogen Idec fragment-based lead discovery strategy. F



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SUNESIS INHIBITOR Perhaps the most selective PDK1 kinase inhibitor reported to date is the pyridinonyl compound 11,31 discovered by Sunesis and Biogen Idec using the fragment-based discovery technology known as tethering with extenders.40 Different from both GlaxoSmithKline and Pfizer compounds, this compound binds to the inactive DFG-out conformation of the PDK1 kinase, a binding mode that could explain its remarkable kinase selectivity described below. First, a mutant version of PDK1 was prepared having a cysteine residue at position 166 (E166C), which is relatively close to the hinge region where ATP binds (Figure 9). Then this protein was reacted with an extender (24) containing sequentially (1) an acrylamide moiety that can react with the introduced cysteine residue, (2) a diaminopyrimidine moiety that is known to bind to the hinge, and (3) a disulfide linker that is reduced subsequently to reveal a free thiol that could react with the disulfide bond of the library compounds. A library of approximately 3000 compounds was screened against this extender containing PDK1 enzyme, identifying pyridinone 25 as a strong hit when an unphosphorylated version of the PDK1 protein was used (Figure 9). Selection of the pyridinone hit was much weaker when phosphorylated PDK1 was used, suggesting that this hit binds to the inactive form of the protein. Replacing the disulfide linkage between the diaminopyrimidine and the pyridinone moieties resulted in compound 26 which inhibited a cascade PDK1-AKT assay with an IC50 of 200 nM (Figure 9). Similar to the screening campaign, this biochemical assay uses unphosphorylated PDK1, which autophosphorylates under the conditions of the assay.31 Medicinal chemistry efforts concentrated on establishing the optimal linker length, exploring purine mimetics to replace the diaminopyrimidine as a hinge-binder, and investigating substituents on the nitrogen of the pyridinone ring. These efforts led to compound 27 which exhibited much higher potency, but also its ethanolamine linker provided an opportunity to perform SAR studies with readily available αamino alcohols (Figure 10). This investigation led to the

discovery of compound 11 which exhibited high potency in the prephosphorylation assay with an IC50 of 2 nM and an EC50 of 400 nM in a cellular assay. An X-ray crystal structure of compound 11 bound to PDK1 confirmed the inhibitor binding to the inactive DFG-out form of the kinase.31 Researchers at Merck profiled compound 11 against large commercial panels of protein kinases as part of a study to identify compounds that could be used as tools in the pharmacological characterization of PDK1.41 Compound 11 showed impressive kinase selectivity, showing greater than 3000-fold selectivity against the 256 kinases tested. Interestingly, cellular studies in PC-3 cells with compound 11 at a 5 μM not only revealed inhibition of AKT Thr-308 and RSK Ser221 but also a slow time-dependent inhibition of the PDK1 autophosphorylation site (p-PDK1 Ser-241), with approximately 70% reduction in phosphorylation at 48 h. Since other PDK1 inhibitors known to bind to the active form of the kinase (DFG-in) do not induce significant dephosphorylation of pPDK1 Ser-241 in cells, the authors speculate that the dephosphorylation observed with compound 11 results from its binding to the inactive form of the kinase (DFG-out), which potentially exposes Ser-241 to solvent, making it vulnerable to phosphatases. The antiproliferative effect of compound 11 was studied on a panel of 17 cancer cell lines representing a variety of tumor types such as breast, colon, lung, brain, and prostate. Similar to results obtained with GlaxoSmithKline compound 8 and Pfizer’s compounds 9 and 10, compound 11 failed to potently inhibit the growth of these cell lines on plastic-attached monolayer cultures (EC50 > 9 μM). This lack of inhibition of monolayer cell growth under standard tissue culture conditions was also observed by genetic PDK1 knockdown experiments. However, compound 11 inhibited the soft agar growth of 4 out of 10 of these cancer cell lines that readily grew in soft agar. Interestingly, all four sensitive cell lines (PC-3, T47D, MDAMB-231, and NCI-H1437) displayed significant compoundmediated PDK1 dephosphorylation of Ser-241, and the authors suggest this inhibition as a potential PD biomarker predictive of efficacy. Consistent with other studies that also demonstrate that PDK1 is required for cancer cell migration and invasion,13 the inhibitory effect of compound 11 on cancer cell growth in soft agar suggests that potent and selective PDK1 inhibitors could potentially be used to inhibit the metastases.41

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PIF POCKET AND PH DOMAIN BINDERS The majority of PDK1 inhibitors reported in the literature are classified as type I ATP-competitive protein kinase inhibitors. However, biochemical and structural data on the mechanism of action of PDK1 revealed two additional sites that could potentially be targeted by small molecules: the PIF pocket and the PH domain. PH Domain Binders. Studies in the literature demonstrate the critical role the PDK1 PH domain plays in the PI3K/AKT pathway and cancer. For instance, mutations in the PDK PH domain that disrupt PIP3 binding result in inhibition of AKT activation in both knock-in mice and homozygous knock-in embryonic stem cells.42,43 In addition, accumulation of PIP3 upon PTEN loss or gain in function of PI3K activity has been detected in approximately 50% of tumors.15,44 On the basis of the structure of 1,3,4,5,6-pentakisphosphate (28), a compound previously reported to inhibit the PI3K/AKT pathway and having proapoptotic45,46 and antitumor activity,47 Falasca and co-workers developed 2-O-benzyl-myo-inositol 1,3,4,5,6-penta-

Figure 10. Lead optimization effort toward 11. G



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except AKT. Several studies have been published demonstrating the PIF pocket as an amenable site for binding of small molecules.49−56 Various screening methods such as virtual screening,49 NMR-based fragment screening,50 and ultrahigh throughput screening (uHTS)51 have all successfully delivered PIF pocket binders, some of these behaving as allosteric activators of PDK1 and inhibitors of the phosphorylation of PDK1 cytosolic substrates that require docking with the PIF pocket for activation (Figure 12). The first report of small molecules binding to the PIF pocket regulatory site in PDK1 was published by Biondi in 2006.49 Compound 29 was discovered through a virtual screening approach targeting the PIF pocket of PDK1 and was shown to increase the intrinsic activity of PDK1 toward Thr-308tide, a polypeptide substrate that comprises the activation loop of AKT,57 with an AC50 of 34 μM. The binding affinity of compound 29 to PDK1 catalytic domain was determined by ITC (Kd = 18 μM). In addition, compound 29 was able to block the PDK1 phosphorylation of its cytosolic substrates S6K and SGK, although high compound concentrations were needed (60−200 μM). A series of PDK1directed mutagenesis experiments, competition experiments with a 24-amino acid polypeptide derived from the HM of PRK2 (PIFtide) using surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) were used to elegantly demonstrate the PIF pocket as the site of binding.49 Subsequently, the same research group obtained an X-ray structure of PDK1 bound to ATP and 30 (PS48), a compound that exhibited a similar AC50 for both the PDK1 full-length and catalytic domain constructs (8 μM).53,54 These crystallographic studies not only confirmed the compounds binding to the PIF pocket but also revealed conformational changes induced allosterically on the ATP binding site hypothesized to activate the kinase. Compounds with similar structures were also discovered by researchers at Pfizer (31) by a NMR-based fragment screening campaign50 and by researchers at Novartis (32) by virtual docking.52 All these compounds have in their structures a carboxylic acid functionality that, when negatively charged, mimics the phosphate group of the phosphorylated HM. In addition, this carboxylate functionality is embedded

kisphosphate (12). This compound exhibited higher efficiency than 28 in inhibiting AKT activation in cell lines characterized by constitutive activation of the PI3K/AKT pathway with a reported sensitivity to 28 (Figure 11).32 Moreover, compound

Figure 11. Falasca’s development of PH domain-targeting inhibitor 12.

12 was able to block AKT phosphorylation on cell lines resistant to 28 including PC3 prostate cells and ASPC1 pancreatic cancer cells. Curiously, compound 12 was able to inhibit the AKT phosphorylation at Ser-473 in addition to phosphorylation at Thr-308. For instance, compound 12 inhibited the AKT phosphorylation at Ser-473 in ovarian cancer cells SKOV-3 at 20 μM after 8 h. Kinase profiling analysis among a panel of approximately 60 kinases screened at 1 μM compound revealed 12 as a highly specific PDK1 inhibitor with an IC50 of 27 nM. Interestingly, 12 also showed some potency against mTOR with an IC50 of 1.3 μM. No inhibition was observed for any of the class I PI3K and AKT isoforms tested. Inhibition of AKT phosphorylation at Thr-308 was also observed in mice treated with 12.32 No data were presented for the inhibitory effect of this compound on other cytosolic substrates of PDK1. Interestingly, inhibition of PDK1 with 12 was able to sensitize cancer cells to the proapoptotic effects of other anticancer compounds, including tamoxifen,32 which is consistent with the reported role of PDK1 inhibition in tamoxifen sensitization.48 PIF Pocket Binders. The PIF pocket is an attractive site for the development of small molecules binders, as it is the docking site required for interaction of PDK1 with all its substrate

Figure 12. PIF pocket binders. H



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effect of their potential PDK1 modulation in cells be determined.

between two aromatic ring hydrophobic moieties, which probably mimics two phenylalanine residues on the HM of the substrate kinases. Interestingly, researchers at Novartis noticed that compounds with similar affinities to the PIF pocket exhibited different levels of kinase activation, likely due to subtle differences in the conformational changes induced by each compound upon binding. Consistent with these findings, alkaloid 33, which was discovered by researchers at Merck by uHTS, did not have an effect on PDK1 enzymatic activity despite its reported binding to the PIF pocket.51 This compound does not have a carboxylate functionality, which will likely result in a distinct binding mode. Recently, a series of dicarboxylate compounds was disclosed by Biondi’s group based on the rationale that a compound with two carboxyl groups might be an improved mimic of the phosphate group on the phosphorylated HM and therefore behave as a more potent activator of PDK1.55,56 Consistent with this hypothesis, compound 34 exhibited a lower AC50 (2 μM) relative to 30 (Figure 12) and, unlike 30, it was able to fully disrupt the interaction between PDK1 and PIFtide. These results were complemented by the X-ray crystal structure of PDK1 in complex with both ATP and compound 34, which showed both carboxylate functionalities engaging in hydrogen bond interactions in the PIF-binding pocket.55,56 Since PIF pocket binding compounds should only block the activation of substrates that requires docking of their respective phosphorylated hydrophobic motif to the PIF pocket, the cellular consequence observed using these compounds might be different from those observed with traditional ATPcompetitive inhibitors. Unfortunately, cellular studies with these carboxylate containing compounds have been hampered because of their poor cellular permeability, probably as a result of the negative charge associated with the carboxylate functionality. Biondi’s group attacked this problem by preparing diester derivative 35 as a cell-permeable prodrug compound of 34 with the hope that once inside the cell, cellular esterases would reveal the active compound 34 (Figure 13).55,56 Cellular

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OUTLOOK Since the initial publication by Berlex Biosciences on compounds 3 and 4,23 many years passed before PDK1 inhibitors were published in the literature with sufficient potency and selectivity to be reliably used in cellular experiments. With the exception of compound 12, which binds to the PH domain of PDK1, the other selective compounds (8, 9, 10, and 11) presented here bind to the active site of the kinase. These medicinal chemistry efforts were driven with X-ray crystallography support, which clearly guided the medicinal chemists’ structure-based design of new analogues. A fragment-based lead discovery strategy was successfully implemented for both GlaxoSmithKline compound 8 and Sunesis compound 11. While GlaxoSmithKline efforts used a more traditional approach by screening a library of hinge binder fragments, Sunesis used the fragment-based discovery technology known as tethering with extenders. The design of their screening assay allowed them to identify compounds that bind to the inactive DFG-out conformation of the PDK1 kinase, a binding mode that could explain its remarkable kinase selectivity. Interestingly, GlaxoSmithKline compound 8 and Pfizer compounds 9 and 10, which bind to the active form of the kinase, seem to achieve their potency and kinase selectivity by specific binding interactions with the PDK1 G-loop. While the reason for this similarity could be just coincidence, it is interesting nonetheless considering that typically the G-loop in protein kinases possesses high flexibility and could pose a challenge for achieving high ligand binding affinity.58 While no selective PDK1 inhibitor has entered the clinic to date, the availability of these potent and highly selective PDK1 inhibitors provides an excellent and unique opportunity to further understand PDK1 biology and potential pharmacological uses of a PDK1 inhibitor. Since most of the research using these compounds has been done in divergent cellular contexts, experiments with these compounds on the same cell lines and same assay conditions should eliminate the ambiguity on the interpretation of the results due to changes in the experimental conditions. GlaxoSmithKline compound 8 and Pfizer compounds 9 and 10 bind to the active form of the PDK1 kinase. Considering that GlaxoSmithKline and Pfizer compounds belong to distinct chemical classes, it is very unlikely that their off-targets effects are exactly the same. Therefore, consistency in cellular consequences using these two compounds would provide confidence that the cellular observations are mainly due to PDK1 inhibition. Sunesis compound 11 binds to the inactive form of PDK1 and induced a slow time-dependent inhibition of the PDK1 autophosphorylation site (p-PDK1 Ser-241). It would be interesting to determine how this deactivation of PDK1 results in phenotypic differences when compared with either Pfizer or GlaxoSmithKline compounds which have not been shown to cause this effect. The work by Merck on compound 11 already presents a correlation between T-loop dephosphorylation and growth inhibition in colony formation assays, suggesting an advantage of this compound over typical ATP-competitive inhibitors. Cellular studies would need to be done to determine whether this time-dependent inhibition of the PDK1 autophosphorylation site will also result in inhibition of the phosphorylation of Thr-308 of AKT even in cells where the PI3K pathway is activated. Falasca’s compound 12 should only inhibit the

Figure 13. Compound 35 as a cell-permeable prodrug compound of 34.

studies with 35 demonstrated inhibition of S6K activity and no effect of the AKT signaling pathway in both HEK293 cells and C2C12 myoblast cells at 50 and 25 μM, respectively.55 The lack of activation of the AKT pathway in cells is consistent with in vitro studies where compound 33 did not show an increase in PDK1 activity when PDKtide, a polypeptide prepared by the joining of PIFtide and Thr-308tide,57 was used as a substrate rather than Thr-308tide. PDKtide is a much better PDK1 substrate than Thr-308tide, and it is probably a better representation of AKT. Although these cellular studies are encouraging, high concentrations of the inhibitor were needed, possibly a result of the compound’s low affinity. Only when more potent PIF pocket binders become available can the true I



Perspective

cancer and other diseases continues to emerge, new opportunities will arise for the use of selective PDK1 inhibitors. The availability of not one but four different classes of potent and selective PDK1 inhibitors to date with distinct binding sites and modes provides an excellent opportunity for researchers in the field. It is up to researchers in both academia and the pharmaceutical industry to take full advantage of this great toolbox of compounds to dissect PDK1 function in cells, predict the pharmacological consequences of PDK1 inhibition, and establish a link to disease treatment in patients.

phosphorylation of Thr-308 of AKT and not the phosphorylation of PDK1 cytosolic substrates, since it binds to the PH domain of PDK1. Therefore, cellular experiments using this compound should provide insight into PIP3 dependent vs PIP3 independent PDK1 mediated signal transduction. It would also be interesting to determine the cellular consequences when combining compound 12 with any of the active site binding compounds. Acquired drug resistance, which negatively affects the clinical success of targeted therapy, is a significant problem where PDK1 inhibitors could be valuable.3 Falasca demonstrated compound 12 to increase the effect of tamoxifen in MCF7breast cancer, consistent with the reported role of PDK1 inhibition in tamoxifen sensitization. Will the other selective PDK1 inhibitors presented here behave similarly to 12? Furthermore, what would be the consequences of combining selective PDK1 inhibitors with other anticancer drugs? There are numerous reports indicating that components of the PI3K/PDK1/AKT pathway control cancer cell resistance to cancer therapies other than tamoxifen.59 For instance, a known mechanism of resistance to the mTORC1 inhibitor rapamycin is linked to its feedback activation of AKT phosphorylation through PI3K and mTORC2.60 The importance of this cross-talk between mTOR and PI3K/AKT signaling pathways has been demonstrated in the clinical setting, and strategies toward the combination of mTOR inhibitors with PI3K inhibitors or the design of molecules that are able to target the kinase activities of both PI3K and mTOR have been pursued.61 Considering the important role of PDK1 in the PI3K/AKT signal transduction pathway, cellular experiments with the available potent and selective PDK1 inhibitors in combination with available mTOR inhibitors should help in the understanding of the complex relationship between AKT and mTOR. Results from these types of experiments are starting to emerge in the literature. As discussed previously, recent work by Alessi provided evidence that phosphorylation of AKT HM at Ser-473, which is mediated by mTORC2, plays a significant role in regulating the phosphorylation of AKT Thr-308. These results suggested that inhibition of Ser-473 with mTOR inhibitors should sensitize AKT activation to PDK1 inhibitors. Consistent with this hypothesis, combination of 1−3 μM 8 with 0.03−01 μM mTOR inhibitor AZD805562 inhibited the phosphorylation of Thr-308 in HCT116, MCF7, and HEL-293 cells to a much greater extent than either inhibitor as a single agent.37 These results are remarkable considering that compound 8 individually was found to be ineffective at inhibiting AKT Thr-308 phosphorylation in response to stimuli that strongly activate PI3K. Considering that compound 12 does possess some mTOR activity and inhibits Ser-473 phosphorylation in cells, it raises the question of whether the effects observed with this compound in cells and in vivo are due to its unique capability to bind to the PH domain or to its dual inhibition of PDK1 and mTOR. The data obtained to date with compound 8 suggest that selective PDK1 inhibition through binding to the active form of the enzyme would be inconsequential in tumors driven by AKT signal transduction. Although a lack of efficacy would be predicted when treating AKT-driven tumors with selective active site PDK1 inhibitors as single agents, the same could not be predicted for tumors driven by AKT-independent pathways where the PDK1 cytosolic substrates may play a crucial role. As knowledge on the role of these PDK1 cytosolic substrates in

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AUTHOR INFORMATION

Corresponding Author

*Phone: 610-917-5889. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biography Jesús R. Medina obtained his Ph.D. in 2000 from the University of Puerto Rico, working with Prof. John Soderquist in the field of organoborane chemistry. Subsequently he joined Clayton Heathcock’s research group at the University of California, Berkeley, as an NIH Postdoctoral Fellow, working on the total synthesis of spongistatin 2. Since 2003 he has been working at GlaxoSmithKline (Upper Providence, PA) as a medicinal chemist in the antibacterial and oncology therapeutic areas.

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ABBREVIATIONS USED PDK1, 3-phosphoinositide-dependent protein kinase 1; PKA, protein kinase A; PKG, protein kinase G; PKC, protein kinase C; T-loop, activation loop; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PH, pleckstrin homology; HM, hydrophobic motif; PIF, 3-phosphoinositidedependent protein kinase 1 interacting fragment; MAPK, mitogen-activated protein kinase; PTEN, phosphatase and tensin homologue deleted on chromosome 10; ES, embryonic stem; LE, ligand efficiency; STD, saturation transfer difference; G-loop, glycine-rich loop; HEK-293, human embryonic kidney 293; IGF1, insulin-like growth factor 1; SGK, serum- and glucocorticoid-induced protein kinase; S6K1, p70 ribosomal S6 kinase 1; NDRG1, N-myc downstream-regulated gene 1; RSK2, p90 ribosomal S6 kinase 2; mTORC2, mammalian target of rapamycin complex 2; uHTS, ultrahigh throughput screening

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REFERENCES

(1) Bunnage, M. E. Getting pharmaceutical R&D back on target. Nat. Chem. Biol. 2011, 7, 335−339. (2) Gashaw, I.; Ellinghaus, P.; Sommer, A.; Asadullah, K. What makes a good drug target? Drug Discovery Today 2011, 16, 1037−1043. (3) Hoelder, S.; Clarke, P. A.; Workman, P. Discovery of small molecule drugs: successes, challenges and opportunities. Mol. Oncol. 2012, 6, 155−176. (4) Quon, K.; Kassner, P. D. RNA interference screening for the discovery of oncology targets. Expert Opin. Ther. Targets 2009, 13, 1027−1035. (5) Cohen, P. Guidelines for the effective use of chemical inhibitors of protein function to understand their roles in cell regulation. Biochem. J. 2010, 425, 53−54. (6) Workman, P.; Collins, I. Probing the probes: fitness factors for small molecule tools. Chem. Biol. 2010, 17, 561−577. (7) Knight, Z. A.; Shokat, K. M. Chemical genetics, where genetics and pharmacology meet. Cell 2007, 128, 425−430. (8) Miyazawa, K. Encountering unpredicted off-target effects of pharmacological inhibitors. J. Biochem. 2011, 150, 1−3. J



Perspective

Romeril, S. P.; Scherzer, D.; Shu, A.; Bobko, M. A.; Chadderton, A. R.; Dumble, M.; Gardiner, C. M.; Gilbert, S.; Liu, Q.; Rabindran, S. K.; Sudakin, V.; Xiang, H.; Brady, P. G.; Campobasso, N.; Ward, P.; Axten, J. M. Structure-based design of potent and selective 3-phosphoinositide-dependent kinase-1 (PDK1) inhibitors. J. Med. Chem. 2011, 54, 1871−1895. (30) Murphy, S. T.; Alton, G.; Bailey, S.; Baxi, S. M.; Burke, B. J.; Chappie, T. A.; Ermolieff, J.; Ferre, R.; Greasley, S.; Hickey, M.; Humphrey, J.; Kablaoui, N.; Kath, J.; Kazmirski, S.; Kraus, M.; Kupchinski, S.; Li, J.; Lingardo, L.; Marx, M. A.; Richter, D.; Tanis, S. P.; Tran, K.; Vernier, W.; Xie, Z.; Yin, M. J.; Yu, X. H. Discovery of novel, potent, and selective inhibitors of 3-phosphoinositide-dependent kinase (PDK1). J. Med. Chem. 2011, 54, 8490−8500. (31) Erlanson, D. A.; Arndt, J. W.; Cancilla, M. T.; Cao, K.; Elling, R. A.; English, N.; Friedman, J.; Hansen, S. K.; Hession, C.; Joseph, I.; Kumaravel, G.; Lee, W. C.; Lind, K. E.; McDowell, R. S.; Miatkowski, K.; Nguyen, C.; Nguyen, T. B.; Park, S.; Pathan, N.; Penny, D. M.; Romanowski, M. J.; Scott, D.; Silvian, L.; Simmons, R. L.; Tangonan, B. T.; Yang, W.; Sun, L. Discovery of a potent and highly selective inhibitor via fragment-based drug discovery. Bioorg. Med. Chem. Lett. 2011, 21, 3078−3083. (32) Falasca, M.; Chiozzotto, D.; Godage, H. Y.; Mazzoletti, M.; Riley, A. M.; Previdi, S.; Potter, B. V. L; Broggini, M.; Maffuci, T. A novel inhibitor of the PI3K/Akt pathway based on the structure of inositol 1,3,4,5,6-pentakisphosphate. Br. J. Cancer 2010, 102, 104−114. (33) Medina, J. R.; Blackledge, C. W.; Heerding, D. A.; Campobasso, N.; Ward, P.; Briand, J.; Wright, L.; Axten, J. M. Aminoindazole PDK1 inhibitors: a case study in fragment-based drug discovery. ACS Med. Chem. Lett. 2010, 1, 439−442. (34) Najafov, A.; Sommer, E. M.; Axten, J. M.; DeYoung, M. P.; Alessi, D. R. Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1. Biochem. J. 2011, 433, 357−369. (35) Alessi, D. R.; Deak, M.; Casamayor, A.; Caudwell, F. B.; Morrice, N.; Norman, D. G.; Gaffney, P.; Reese, C. B.; MacDougall, C. N.; Harbison, D.; Ashworth, A.; Bownes, M. 3-Phosphoinositidedependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr. Biol. 1997, 7, 776−789. (36) Alessi, D. R.; Kozlowski, M. T.; Weng, Q. P.; Morrice, N.; Avruch, J. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr. Biol. 1998, 8, 69−81. (37) Najafov, A.; Shpiro, N.; Alessi, D. R. Akt is efficiently activated by PIF-pocket- and PtdIns(3,4,5)P3-dependent mechanism leading to resistance to PDK1 inhibitors. Biochem. J. 2012, 448, 285−295. (38) Ellwood-Yen, K.; Keilhack, H.; Kunii, K.; Dolinski, B.; Connor, Y.; Hu, K.; Nagashima, K.; O’Hare, E.; Erkul, Y.; Bacco, A. D.; Gargano, D.; Shomer, N. H.; Angagaw, M.; Leccese, E.; Andrade, P.; Hurd, M.; Shin, M. K.; Vogt, T. F.; Nothrup, A.; Bobkova, E. V.; Kasibhatla, S.; Bronson, R. T.; Scott, M. L.; Draetta, G.; Richon, V.; Kohl, N.; Blume-Jensen, P.; Andersen, J. N.; Kraus, M. PDK1 attenuation fails to prevent tumor formation in PTEN-deficient transgenic mouse models. Cancer Res. 2011, 71, 3052−3065. (39) Axten, J. M.; Medina, J. R.; Shu, A.; Li, W.; Feng, Y.; DeYoung, M. P.; Gardiner, C. M.; Chadderton, A. R.; Dumble, M.; Liu, Q.; Rabindran, S. K. GSK2334470: A Potent and Highly Selective Inhibitor of PDK1. Abstracts of Papers, 101st Annual Meeting of AACR, Washington DC, April 17−21, 2010; AACR: Philadelphia, PA, 2010; Abstract LB-116. (40) Erlanson, D. A.; Lam, J. W.; Wiesmann, C.; Luong, T. N.; Simmons, R. L.; DeLano, W. L.; Choong, I. C.; Burdett, M. T.; Flanagan, W. M.; Lee, D.; Gordon, E. M.; O’Brien, T. In situ assembly of enzyme inhibitors using extended tethering. Nat. Biotechnol. 2003, 21, 308−314. (41) Nagashima, K.; Shumway, S. D.; Sathyanarayanan, S.; Chen, A. H.; Dolinski, B.; Xu, Y.; Keilhack, H.; Nguyen, T.; Wiznerowicz, M.; Li, L.; Lutterbach, B. A.; Chi, A.; Paweletz, C.; Allison, T.; Yan, Y.; Munshi, S. K.; Klippel, A.; Kraus, M.; Bobkova, E. V.; Deshmukh, S.; Xu, Z.; Mueller, U.; Szewczak, A. A.; Pan, B. S.; Richon, V.; Pollock,

(9) Zhang, J.; Yang, P. L.; Gray, N. S. Targeting cancer with small molecule kinase inhibitors. Nat. Rev. Cancer 2009, 9, 28−39. (10) Bain, J.; Plater, L.; Elliot, M.; Shpiro, N.; Hastie, C. J.; McLauchlan, H.; Klevernic, I.; Arthur, J. S. C.; Alessi, D. R.; Cohen, P. The selectivity of protein kinase inhibitors: a further update. Biochem. J. 2007, 408, 297−315. (11) Liao, J. J-L. Molecular recognition of protein binding pockets for design of potent and selective kinase inhibitors. J. Med. Chem. 2007, 50, 409−424. (12) Barile, E.; De, S. K.; Pellecchia, M. PDK1 inhibitors. Pharm. Pat. Analyst 2012, 1, 145−163. (13) Raimondi, C.; Falasca, M. Targeting PDK1 in Cancer. Curr. Med. Chem. 2011, 18, 2763−2769. (14) Peifer, C.; Alessi, D. R. Small-molecule inhibitors of PDK1. ChemMedChem 2008, 3, 1810−1838. (15) Pearce, L. R.; Komander, D.; Alessi, D. R. The nuts and bolts of AGC protein kinases. Nat. Rev. Mol. Cell Biol. 2010, 9−22. (16) Alessi, D. R.; James, S. R.; Downes, C. P.; Holmes, A. B.; Gaffney, P. R.; Reese, C. B.; Cohen, P. Characterization of a 3phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr. Biol. 1997, 7, 261−269. (17) Stokoe, D.; Stephens, L. R.; Copeland, T.; Gaffney, P. R. J.; Reese, C. B.; Painter, G. F.; Holmes, A. B.; McCormic, F.; Hawkins, P. T. Dual role of phosphatidylinositol-3,4,5-triphosphate in the activation of protein kinase B. Science 1997, 277, 567−570. (18) Milburn, C. C.; Deak, M.; Kelly, S. M.; Price, N. C.; Alessi, D. R.; Van Aalten, D. M. Binding of phosphatidylinositol 3,4,5triphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change. Biochem. J. 2003, 375, 531−638. (19) Biondi, R. M.; Kieloch, A.; Currie, R. A.; Deak, M.; Alessi, D. R. The PIF-binding pocket in PDK1 is essential for activation of S6K and SGC, but not PKB. EMBO J. 2001, 20, 4380−4390. (20) Mora, A.; Komander, D.; Van Aalten, D. M. F.; Alessi, D. R. PDK1, the master regulator of AGC kinase signal transduction. Semin. Cell Dev. Biol. 2004, 15, 161−170. (21) Bayascas, J. R.; Leslie, N. R.; Parsons, R.; Fleming, S.; Alessi, D. R. Hypomorphic mutation of PDK1 suppresses tumorigenesis in PTEN(+/−) mice. Curr. Biol. 2005, 15, 1839−1846. (22) Zhu, J.; Huang, J. W.; Tseng, P. H.; Yang, Y. T.; Fowble, J.; Shiau, C. W.; Shaw, Y. J.; Kulp, S. K.; Chen, C. S. From the cyclooxygenase-2 inhibitor celecoxib to a novel class of 3phosphoinisitide-dependent protein kinase-1 inhibitors. Cancer Res. 2004, 64, 4309−4318. (23) Feldman, R. I.; Wu, J. M.; Polokoff, M. A.; Kochanny, M. J.; Dinter, H.; Zhu, D.; Biroc, S. L.; Alicke, B.; Bryant, J.; Yuan, S.; Buckman, B. O.; Lentz, D.; Ferrer, M.; Whitlow, M.; Adler, M.; Finster, S.; Chang, Z.; Arnaiz, D. O. Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1. J. Biol. Chem. 2005, 280, 19867−19874. (24) Tamgüney, T.; Zhang, C.; Fiedler, D.; Shokat, K.; Stokoe, D. Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells. Exp. Cell Res. 2008, 314, 2299−2312. (25) Poulsen, A.; Blanchard, S.; Soh, C. K.; Lee, C.; Williams, M.; Wang, H.; Dymock, B. Structure-based design of PDK1 inhibitors. Bioorg. Med. Chem. Lett. 2012, 22, 305−307. (26) Yang, W.; AbdulHameed, M. D. M.; Hamza, A.; Zhan, C. G. New inhibitor of 3-phosphoinositide dependent protein kinase-1 identified from virtual screening. Bioorg. Med. Chem. Lett. 2012, 22, 1629−1632. (27) Johnson, M. C.; Hu, Q.; Lingardo, L.; Ferre, R. A.; Greasley, S.; Yan, J.; Kath, J.; Chen, P.; Ermolieff, J.; Alton, G. Novel isoquinolone PDK1 inhibitors discovered through fragment-based lead discovery. J. Comput.-Aided Mol. Des. 2011, 25, 689−698. (28) Angiolini, M.; Banfi, P.; Casale, E.; Casuscelli, F.; Fiorelli, C.; Saccardo, M. B.; Silvagni, M.; Zuccotto, F. Structure-based optimization of potent PDK1 inhibitors. Bioorg. Med. Chem. Lett. 2010, 20, 4095−4099. (29) Medina, J. R.; Becker, C. J.; Blackledge, C. W.; Duquenne, C.; Feng, Y.; Grant, S. W.; Heerding, D.; Li, W. H.; Miller, W. H.; K



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kinase-1: development and prodrug concept. J. Med. Chem. 2012, 55, 9817−9830. (57) Biondi, R. M.; Cheung, P. C.; Casamayor, A.; Deak, M.; Currie, R. A.; Alessi, D. R. Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA. EMBO J. 2000, 19, 979−988. (58) Liao, J. J.-L. Molecular recognition of protein kinase binding pockets for design of potent and selective kinase inhibitors. J. Med. Chem. 2007, 50, 409−424. (59) Falasca, M. PI3K/Akt signalling pathway specific inhibitors: a novel strategy to sensitize cancer cells to anti-cancer drugs. Curr. Pharm. Des. 2010, 16, 1410−1416. (60) Mavrommati, I.; Maffucci, T. mTOR inhibitors: facing new challenges ahead. Curr. Med. Chem. 2011, 18, 2743−2762. (61) Willems, L.; Tamburini, J.; Chapuis, N.; Lacombe, C.; Mayeux, P.; Bouscary, D. PI3K and mTOR signaling pathways in cancer: new data on targeted therapies. Curr. Oncol. Rep. 2012, 14, 129−138. (62) Chresta, C. M.; Davies, B. R.; Hickson, I.; Harding, T.; Cosulich, S.; Critchlow, S. E.; Vincent, J. P.; Ellston, R.; Jones, D.; Sini, P.; James, D.; Howard, Z.; Dudley, P.; Hughes, G.; Smith, L.; Maguire, S.; Hummerson, M.; Malagu, K.; Menear, K.; Jenkins, R.; Jacobsen, M.; Smith, G. C.; Guichard, S.; Pass, M. AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. Cancer Res. 2010, 70, 288−298.

R.; Blume-Jensen, P.; Northrup, A.; Andersen, J. N. Genetic and pharmacological inhibition of PDK1 in cancer cells. Characterization of a selective allosteric kinase inhibitor. J. Biol. Chem. 2011, 286, 6433−6448. (42) Bayascas, J. R.; Wullschleger, S.; Sakamoto, K.; García-Martínez, J. M.; Clacher, C.; Komander, D.; van Aalten, D. M.; Boini, K. M.; Lang, F.; Lipina, C.; Logie, L.; Sutherland, C.; Chudek, J. A.; van Diepen, J. A.; Voshol, P. J.; Lucocq, J. M.; Alessi, D. R. Mutation of the PDK1 PH domain inhibits protein kinase B/Akt, leading to small size and insulin resistance. Mol. Cell. Biol. 2008, 28, 3258−3272. (43) McManus, E. J.; Collins, B. J.; Ashby, P. R.; Prescott, A. R.; Murray-Tait, V.; Armit, L. J.; Arthur, J. S.; Alessi, D. R. The in vivo role of PtIns(3,4,5)P3 binding to PDK1 PH domain by knockin mutation. EMBO J. 2004, 23, 2071−2082. (44) Carracedo, A.; Pandolfi, P. P. The PTEN-PI3K pathway: of feedbacks and cross-talks. Oncogene 2008, 27, 5527−5541. (45) Piccolo, E.; Vignati, S.; Maffuci, T.; Innominato, P. F.; Riley, A. M.; Potter, B. V.; Pandolfi, P. P.; Broggini, M.; Iacobelli, S.; Innocenti, P.; Falasca, M. Inositol pentakisphosphate promotes apoptosis through the PI 3-K/Akt pathway. Oncogene 2004, 23, 1754−1765. (46) Razzini, G.; Berrie, C. P.; Vignati, S.; Broggini, M.; Mascetta, G.; Brancaccio, A.; Falasca, M. Novel functional PI 3-kinase antagonists inhibit cell growth and tumorigenicity in human cancer cell lines. FASEB J. 2000, 14, 1179−1187. (47) Maffucci, T.; Falasca, M. Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositideprotein co-operative mechanism. FEBS Lett. 2001, 506, 173−179. (48) Iorns, E.; Lord, C. J.; Ashworth, A. Parallel RNAi and compound screens identify the PDK1 pathway as a target for tamoxifen sensitization. Biochem. J. 2009, 417, 361−370. (49) Engel, M.; Hindie, V.; Lopez-Garcia, L. A.; Stroba, A.; Schaeffer, F.; Adrian, I.; Imig, J.; Idrissova, L.; Nastainczyk, W.; Zeuzem, S.; Alzari, P. M.; Hartmann, R. W.; Piiper, A.; Biondi, R, M. Allosteric activation of the protein kinase PDK1 with low molecular weight compounds. EMBO J. 2006, 25, 5469−5480. (50) Stockman, B. J.; Kothe, M.; Kohls, D.; Weibley, L.; Connolly, B. J.; Sheils, A. L.; Cao, Q.; Cheng, A. C.; Yang, L.; Kamath, A. V.; Ding, Y. H.; Charlton, M. E. Identification of allosteric PIF-pocket ligands for PDK1 using NMR-based fragment screening and 1H-15N TROSY experiments. Chem. Biol. Drug Des. 2009, 73, 179−188. (51) Bobkova, E. V.; Weber, M. J.; Xu, Z.; Zhang, Y. L.; Jung, J.; Blume-Jensen, P.; Northrup, A.; Kunapuli, P.; Andersen, J. N.; Kariv, I. Discovery of PDK1 kinase inhibitors with a novel mechanism of action by ultrahigh throughput screening. J. Biol. Chem. 2010, 285, 18838− 18846. (52) Wei, L.; Gao, X.; Warne, R.; Hao, X.; Bussiere, D.; Gu, X.; Uno, T.; Liu, Y. Design and synthesis of benzoazepin-2-one analogs as allosteric binders targeting the PIF pocket of PDK1. Bioorg. Med. Chem. Lett. 2010, 20, 3897−3902. (53) Hindie, V.; Stroba, A.; Zhang, H.; Lopez-Garcia, L. A.; Idrissova, L.; Zeuzem, S.; Hirschberg, D.; Schaeffer, F.; Jørgensen, T. J. D.; Engel, M.; Alzari, P. M.; Biondi, R. M. Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1. Nat. Chem. Biol. 2009, 5, 758−764. (54) Stroba, A.; Schaeffer, F.; Hindie, V.; Lopez-Garcia, L.; Adrian, I.; Fröhner, W.; Hartman, R. W.; Biondi, R. M.; Engel, M. 3,5Diphenylpent-2-enoic acids as allosteric activators of the protein kinase PDK1: structure−activity relationships and thermodynamic characterization of binding as paradigms for PIF-binding pockettargeting compounds. J. Med. Chem. 2009, 52, 4683−4693. (55) Busschots, K.; Lopez-Garcia, L. A.; Lammi, C.; Stroba, A.; Zeuzem, S.; Piiper, A.; Alzari, P. M.; Neimanis, S.; Arencibia, J. M.; Engel, M.; Schulze, J. O.; Biondi, R. M. Substrate-selective inhibition of protein kinase PDK1 by small compounds that bind to the PIF-pocket allosteric docking site. Chem. Biol. 2012, 19, 1152−1163. (56) Wilhelm, A.; Lopez-Garcia, L. A.; Busschots, K.; Fröhner, W.; Maurer, F.; Boettcher, S.; Zhang, H.; Schulze, J. O.; Biondi, R. M.; Engel, M. 2-(3-Oxo-1,3-diphenylpropyl)malonic acids as potent allosteric ligands of the PIF pocket of phosphoinositide-dependent L


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