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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
Pharmacopeia Drug Discovery Inc., Princeton, New Jersey
Received for publication
October 24, 2005
Accepted
May 12, 2006.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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kinase and the lipopolysaccharide (LPS)-induced increase in tumor necrosis factor (TNF) levels in cell media of human monocytes with IC50 values of 1 µM. The screening data revealed a preferred synthon, 3-amino-4-methyl benzamide, which is critical for the activity against p38. This synthon appeared almost exclusively in screening hits including PS200981, and slight variations of this synthon including 3-amino benzamide and 2-amino-4-methyl benzamide also contained in the library were inactive. PS200981 is equally potent against the and 
forms of p38 but did not inhibit p38
and is >25-fold selective versus a panel of other kinases. PS200981 inhibited the LPS-induced increase in TNF levels when administered at 30 mg/kg to mice. Selectivity and in vivo activity of this class of p38 inhibitors was further demonstrated by PS166276 [(R)-3-(4-(isobutyl(methyl)-amino)-6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)-4-methylbenzamide], a highly structurally related but more potent and less cytotoxic inhibitor, in several intracellular signaling assays, and in LPS-challenged mice. Overall, this novel class of p38 inhibitors is potent, active in vitro and in vivo, and is highly selective.
and interleukin 1, endotoxin, UV irradiation, heat, and osmotic shock (Pearson et al., 2001
). Activation of p38 requires dual phosphorylation by upstream MAP kinase kinases (MKKs) 3 and 6 on a threonine and a tyrosine within a Thr-Gly-Tyr motif, characteristic of p38 kinases (Raingeaud et al., 1996). Once activated, p38 kinases phosphorylate and activate other kinases and transcription factors leading to increases or decreases in the expression of certain target genes, such as interleukin-1 and TNF (Lee et al., 1994).
The up-regulation by activated p38 kinase of cytokine production, predominantly TNF and interleukin-1, is a hallmark of inflammation associated with a wide variety of diseases such as rheumatoid arthritis, endotoxic shock, inflammatory bowel disease, multiple sclerosis, psoriasis, and others (Henry et al., 1999; Salituro et al., 1999). Several biological agents that sequester TNF or inhibit the action of interleukin-1, including monoclonal antibody to TNF (infliximab and Adalimumab) (Onrust and Lamb, 1998; Furst et al., 2003), soluble TNF receptor-Fc fusion protein (Etanercept) (Jarvis and Faulds, 1999), or the interleukin-1 receptor antagonist (anakinra) (Cvetkovic and Keating, 2002) have been used to treat patients with chronic inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, and psoriasis. Although these agents are efficacious, they are costly and limited by route of administration. Thus, a need exists for small-molecule-based therapeutics that can be administered orally and at lower cost to both patient and health care systems.
The challenge in identification of kinase inhibitors for use in chronic disease is the potential for off-target effects at other kinases that cause issues with the safety profile. This is due largely to the general conservation in the ATP-binding site of kinases and the specific homology across related members of specific kinase subfamilies. The MAP kinase family consists of three subfamilies that include the extracellular signal-regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs), and p38 kinases. Molecular cloning studies have led to the identification of four p38 isoforms: p38, p38, p38, and p38
. These isoforms differ in tissue distribution, substrate preference, and activation modes (Goedert et al., 1997; Wang et al., 1997). Among these four isoforms, p38 is the best characterized and perhaps the most physiologically relevant p38 isoform involved in inflammatory responses (Allen et al., 2000).
In the past few years, a number of small-molecule p38 inhibitors have been shown to block the production of TNF and interleukin-1 (Cirillo et al., 2002; Jackson and Bullington, 2002), and some of these have advanced to clinical studies (Kumar et al., 2003). Concerns about off-target effects, however, have been raised. For example, VX-745 [5-(2,6-dichlorophenyl)-2-(phenylthio)-6H-pyrimido[1,6-b]pyridazin-6-one], an ATP-competitive p38 inhibitor, advanced to clinical trials but was withdrawn following issues with central nervous system toxicity and BIRB-796 (doramapimod), an allosteric p38 inhibitor with a slow off-rate (Kumar et al., 2003), also advanced to clinical trials and was last reported in phase III. In this article, we report the discovery of a novel and highly selective class of p38 inhibitors that demonstrate a strong selection for the methylanilino amide synthon, indicating a critical interaction with p38.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Tissue Culture. Human monocytic (THP-1) cells were obtained from American Type Culture Collection (Manassas, VA), maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, and split twice a week. F7 cells were kindly provided by Dr. Hodaka Fujii, University of Tokyo (Hatakeyama et al., 1989). F7 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum plus interleukin-2.
Generation of p38 Kinases. Human p38 cDNA was amplified from human liver Quick-Clone cDNA (BD Biosciences), and human p38 and p38 cDNAs were amplified from human brain quick-clone cDNA using polymerase chain reaction technology. p38 cDNAs were subcloned in the prokaryotic expression vector pGEX, which consisted of a glutathione S-transferase (GST) sequence at the amino-terminal region (Amersham Biosciences, Piscataway, NJ). The expression vectors containing p38, , and were transformed into the BL21 (DE3) strain of Escherichia coli. Expression of GST-p38 fusion proteins was induced in the presence of isopropyl -D-1-thiogalacto-pyranoside. GST-p38 fusion proteins were purified from bacterial pellets using affinity chromatography (Amersham Biosciences). p38, , and isozymes were activated using constitutively active MKK6.
p38 Kinase Assay. The p38 kinase screen was performed in a 96-well filter mat assay format with myelin basic protein as the substrate. p38 was preincubated with test compounds for 10 min. The reaction was initiated by adding substrate mix containing [-33P]ATP and protein substrate. After a 45-min incubation, the reaction was terminated by adding EDTA (40 mM final), the protein substrate was then harvested onto filter mats, and [-33P]ATP was removed using a Skatron Micro96 Cell Harvester (Molecular Devices, Sunnyvale CA). -33P-phosphorylated protein substrate was detected using a Microbeta scintillation counter (PerkinElmer, Wellesley, MA). The final concentrations of reagents in the reactions were 10 nM p38, 34 µg/ml myelin basic protein, 50 mM Tris, pH 7.5, 10 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 1 µM ATP, 3 nM [-33P]ATP, and 0.3% dimethyl sulfoxide.
Kinase Selectivity Assays. Different assay formats were used in the evaluation of selectivity for p38 against a panel of non-p38 kinases. To ensure similar sensitivity across the different kinase assays, the ATP concentration was adjusted to equal or below Km for each kinase.
The filter mat protocol described under Materials and Methods was used to determine selectivity against ERK1, ERK2, JNK11, JNK22, Mapkap K2, and protein kinase A. Myelin basic protein was used as the substrate for ERK1, ERK2, and Mapkap-K2; histone was used as the substrate for protein kinase A; and ATF-2 was used as the substrate for JNK11 and JNK22.
A time-resolved fluorescence assay was used to determine selectivity against Src and ZAP-70. In brief, the protein substrate, poly Glu-Tyr (25 µg/ml) for Src and cdb3 (10 µg/ml) for ZAP-70, was immobilized in black 384-well plates. Tyrosine kinases were preincubated with compounds for 10 min, and the reactions were initiated by adding ATP. The plates were incubated for 45 min at room temperature. The reaction mixes were removed, and the plates were washed twice with Tris-buffered saline. Europium-labeled antiphosphotyrosine antibody (75 ng/ml) was added to the plates and incubated for 1 h at room temperature. The plates were washed five times with Tris-buffered saline. The signal from bound Europium-labeled antiphosphotyrosine antibody was measured using Victor (PerkinElmer) in the presence of enhancement solution.
A homogeneous time-resolved fluorescence assay was used to determine selectivity against Abl and Tie2. Biotinylated poly Glu-Ala-Tyr was used as the substrate. Kinases were preincubated with compounds for 10 min. The reaction was initiated by adding substrate solution containing biotinylated poly Glu-Ala-Tyr and ATP. After incubation for 1 h at room temperature, the reaction was terminated by adding stop/detection solution containing EDTA (3 mM final), streptavidin-Cy5 (1.5 µg/ml final), and Eu-PT66 (0.2 µg/ml final). The plate was read by Victor V (PerkinElmer) at both 615 and 665 nm. The signal is analyzed using the ratio of 665- to 615-nm fluorescence signal. Selectivity against PKB, RSK2, and CaMKIV was assessed using IMAP assays (Molecular Devices) according to the manufacturer's protocols. Kinase assays for Aurora-A, BTK, CDK1/cyclin B, CHK1, CK1, CK2, CSK, EGFR, EphB4, FGFR3, Flt3, GSK3, Flt1, IGF-1R, IKK, KDR, Lck, Met, NEK2, PAK4, PDK1, PKC, PKC
, and ROCK-II were performed using the Kinase Profiler Assay Protocols from Upstate Biotechnology Inc. and validated with staurosporine.
LPS-Induced TNF Production in THP-1 Cells. THP-1 cells were seeded in 96-well tissue culture plates. Test compounds or vehicle (1% dimethyl sulfoxide final) were added to cells followed by the addition of LPS (1 µg/ml final). Plates were incubated overnight at 37°C and 5% CO2. TNF in the medium was measured using a sandwich immunoassay. TNF in the supernatant was immobilized by an anti-human TNF antibody (R&D, Minneapolis, MN; no. MAB610), which was precoated in high-binding ELISA immunoassay plates. Immobilized TNF was recognized by a biotinylated anti-human TNF polyclonal antibody (R&D; no. BAF210). Streptavidin conjugated to horseradish peroxidase was used in the ELISA, and the activity of peroxidase was quantified using a peroxide substrate kit (Pierce Biotechnology, Rockford, IL).
Cytotoxicity Assay. Cytotoxicity was evaluated in THP-1 cells. THP-1 cells were harvested at log phase, resuspended in fresh medium, and seeded in 96-well plates (30,000 cells/well). Compounds were serially diluted and added to cells. After 48-h incubation, cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reagent and assayed (Promega, Madison, WI) according to the manufacturer's instructions.
Mapkap-K2 Immune Complex Kinase Assay. THP-1 cells were treated as described above. Cells were lysed in radioimmunoprecipitation assay buffer (50 mM Tris, pH 7.5, 1% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium vanadate, 1 mM nitrophenylphosphate, 5 mM benzamidine, 0.2 µM calyculin A, 2 mM phenylmethylsulfonyl fluoride, and 10 mg/ml aprotinin). Lysates were cleared by centrifugation (10,000g, 10 min) following incubation with antibody recognizing Mapkap-K2 for 1 h at 4°C and protein G-Sepharose for an additional 2 h at 4°C. The precipitates were washed three times with cold wash buffer (0.25 M Tris, pH 7.5, 0.1 M NaCl). The immune complexes were resuspended in 40 µl of kinase assay buffer containing 10 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 10 µM ATP, 5 µCi of [-32P]ATP, and 2 µg of hsp27 and incubated for 30 min at room temperature. Reactions were stopped by adding 40 µl of 2x sample buffer and boiling for 3 min. The samples were subjected to SDS-polyacrylamide gel electrophoresis (12%), and the gels were dried. The dried gels were exposed to X-ray films.
Western Blot Analysis. Total cell lysates from equivalent cell numbers were separated using SDS-polyacrylamide gel electrophoresis (10%) under reducing conditions. The proteins were transferred electrophoretically onto polyvinylidene fluoride membrane (Immobilon P; Millipore, Billerica, MA). The membranes were blocked with 1% bovine serum albumin in PBS. The membranes were incubated first with primary antibody (1 µg/ml) in 1% bovine serum albumin/PBS and then with secondary antibody conjugated with peroxidase in 5% nonfat dry milk/PBS. The immunocomplexes were detected using an enhanced chemiluminescence kit (Amersham Biosciences).
Inhibition of TNF Release in Mice. Female BALB/c mice, weighing approximately 20 g, were used to evaluate PS200981 and PS166276 on TNF release in vivo. PS200981 and PS166276 were dissolved in 5% ethanol, 5% Tween 80, and 90% water. Mice were dosed s.c. with PS200981, PS166276, PBS, or vehicle 30 min prior to LPS challenge. LPS (0.2 ml of LPS suspended at 10 µg/ml in saline) was injected in mice i.v. Blood samples were obtained 60 min after LPS injection, and serum was separated by centrifugation. Levels of TNF were measured using an ELISA kit (R&D).
Synthesis of PS200981 and PS166276. The structures of PS200981 and PS166276 are shown in Fig. 1. A solution phase synthesis for this series of p38 inhibitors, including PS200981 and PS166276, was described previously (Leftheris et al., 2004).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Inhibitors. A total of 41 Encoded Combinatorial Libraries on Polymeric Support libraries, comprising 2.1 million compounds, were screened. Compounds were arrayed in plates at 10 to 20 compounds per well for the initial survey. Putative active libraries were then arrayed as a single compound per well at a 3-fold redundancy. The structures in the active wells were identified through a proprietary decoding process, followed by compound resynthesis to confirm activity (Diller and Hobbs, 2004).
A combinatorial library was found to contain numerous active compounds. This library contains 
180,000 members and was constructed through a linear, four-combinatorial step synthesis (31 R1 x 63 R2 x 3 R3 x 31 R4) based on a triaminotriazine structure. The frequency of individual combinatorial synthons present within the hits is shown in Fig. 2. Of 63 different R2 synthons used in the construction of this combinatorial library, 63 of 69 screening hits contain synthon R2 39, indicating an almost exclusive preference for synthon R2 39, which corresponds to a methylanilino amide. A somewhat broader preference is also evident within the R4 components, whereas no preference is observed in the R1 and R3 components.
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and TNF Production by PS200981. PS200981, a triaminotriazine aniline amide compound that showed inhibition of p38 in the screen, was further tested in a panel of in vitro kinase and cell-based assays. Figure 3A shows the inhibition of p38 activity by PS200981 in a dose-dependent manner. The IC50 value was 1.0 µM, in which the ATP concentration was 1 µM in the assay. Evaluation of the inhibition of TNF production by PS200981 in cells was accomplished using LPS induction of TNF in human monocytic THP-1 cells. As seen in Fig. 3B, PS200981 inhibited TNF production with an IC50 value of 1.0 µM.
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PS200981 Is an ATP Competitive Inhibitor. Kinetic experiments were conducted to determine whether PS200981 was an ATP competitive inhibitor. The velocity of p38 was determined in the presence of various concentrations of ATP in combination with different concentrations of PS200981. Figure 4 shows Lineweaver-Burke plots of the competition of PS200981 with ATP in p38 kinase activity. These data are consistent with a competitive mechanism of action for PS200981. The Ki of 1 µM is further consistent with the IC50 value of 1.0 µM assayed in the presence of 1 µM ATP. The value of Km[ATP] in this reaction is 27 µM, which is consistent with the results reported previously (Frantz et al., 1998).
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Kinase Selectivity. The p38 family is one of three related kinase families, the others being ERK and JNK. Moreover, p38 kinase exists as four distinct isoforms (, , , and ). Thus, a well understood kinase selectivity profile is essential for use of p38 kinase inhibitors in chronic diseases. To this end, the kinase selectivity of the hit series was examined against a variety of recombinant kinases. PS200981 exhibits equipotent inhibitory activity against and isoforms and no activity against the isoform (Table 1). It is of interest to note that several structurally diverse p38 kinase inhibitors, such as VX-745, SB203580 [4-[5-(4-fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-1H-imidazol-4-yl]-pyridine], RWJ67657 [4-(4-fluorophenyl)-2-(4-hydroxy-1-butynyl)-1-(3-phenylpropyl)-5-(4-pyridyl)imidazole], BIRB-796, and RPR200756A [4-[[trans-2-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-5-methyl-1,3-dioxan-5-yl]carbonyl]-morpholine], inhibit the p38 and p38 isoforms but not the and isoforms (Kumar et al., 1997; Wadsworth et al., 1999; McLay et al., 2001; Pargellis et al., 2002; Natarajan et al., 2003). As shown in Table 1, neither PS200981 nor PS166276 inhibited other MAP kinase family members, such as ERK1, ERK2, JNK11, and JNK22. Furthermore, these compounds showed greater than 50-fold selectivity versus 31 other kinases including Mapkap K2, a downstream kinase of p38, whereas it only weakly inhibited Abl and Tie2.
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To assess kinase selectivity at the cellular level, PS166276 (Fig. 1), which has the same critical triaminotriazine aniline amide substitution at R2, but has different R1 and R3 groups compared with PS200981, was used. PS166276 has an IC50 of 28 nM at p38 kinase and 170 nM in the THP-1 TNF assay (Fig. 3). Similar to PS200981, PS166276 is also highly selective for p38 and ; it is greater than 1000-fold selective versus a panel of 38 kinases (Table 1). Although PS200981 and PS166276 are structurally similar, both compounds exhibit different cytotoxicity. In THP-1 cells, PS200981 was cytotoxic at concentrations greater than 10 µM, whereas PS166276 showed no cytotoxicity up to 100 µM, the highest concentration used in the assay (data not shown). Because PS166276 is highly structurally related, 35-fold more potent, and 10-fold less cytotoxic than PS200981, it was more suitable for assessing kinase selectivity in intact cells. These assays monitored the effects of PS166276 on kinase-mediated signal transduction cascades, including the MAP kinase pathways (p38, ERK, and JNK), IKK, and Jak/Stat pathways.
The serine/threonine kinase Mapkap K2, a downstream kinase of p38, is phosphorylated and activated by p38 (Kotlyarov et al., 1999). Thus, inhibition of p38-mediated signal transduction was evaluated by examining the effects of compound treatment on Mapkap K2 activation by p38. THP-1 human monocytic cells were pretreated with PS166276 prior to challenge with LPS. Mapkap K2 was immunoprecipitated from cell lysates using an antibody. Kinase activity of Mapkap K2 in the immunocomplexes was determined using hsp27 as a substrate. LPS robustly increased Mapkap K2 activity, which was suppressed in a dose-dependent manner by PS166276 (Fig. 5A). Higher compound concentrations (10 and 3 µM) completely abrogated Mapkap K2 activation, whereas at 1 and 0.3 µM, partial inhibition was observed. An IC50 value of 200 nM was estimated, which is consistent with the potency observed in the LPS-induced TNF- production assay (Fig. 3). PS166276 did not inhibit Mapkap K2 kinase activity when added directly to immunoprecipitated MK2 (data not shown).
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Similar to the p38 kinase pathway, JNK is activated by cellular stress via a signal transduction cascade that involves MKK4/7. Phosphorylation of JNK at Thr183 and Tyr185 results in catalytic activation. The substrates for JNK2 include transcription factors such as c-Jun and ATF-2. LPS treatment of THP-1 cells resulted in JNK activation, which was detected as phosphorylation of ATF-2 using an antiphospho-ATF-2 antibody (Fig. 5C). PS166276 had no effect on ATF-2 phosphorylation at concentrations of up to 30 µM, suggesting that the compound does not interfere with JNK signaling components.
The IKK pathway can be activated by endotoxin or proinflammatory cytokines. The IKK isozymes activate nuclear factor-
B-mediated signal transduction by phosphorylation of IB, which subsequently undergoes proteolytic degradation. Degradation of IB was monitored by immunoblot to determine the effects of PS166276 on the IKK pathway. As shown in Fig. 5B, LPS promptly induced degradation of IB in THP1 cells. No effect was observed at compound concentrations of up to 30 µM, indicating that the compound effects on TNF and the expression of other proinflammatory cytokines are not mediated through IKK inhibition. Similar results were observed in the TNF-stimulated IB degradation in U937 cells (data not shown).
Similar analyses were applied to the Jak2 and Jak3 pathways using the mouse F7 pre-B lymphocyte cell line. These cells are dually responsive to interleukin-2 (Jak1/3-dependent) and interleukin-3 (Jak2-dependent). Following interleukin-2 or -3 binding to its corresponding receptor, Jak2/3 kinases are activated by phosphorylation and are capable of catalyzing phosphoryl transfer to Stat5. The effects of PS166276 on Stat5 phosphorylation are shown in Fig. 5, E and F. PS166276 had no effect on the interleukin-2 and -3-induced Stat5 phosphorylation. Taken together, these results indicate that this series of compounds is highly selective for p38 versus other protein kinases.
PS200981 and PS166276 Inhibit LPS-Induced TNF Production in Mice. Although PS200981 was identified directly from high throughput screening, its efficacy was demonstrated in vivo. Because PS200981 has low cellular permeability (data not shown), suggesting low oral absorption in animals, compounds were administered to mice s.c. As seen in Fig. 6A, PS200981, at 30 mg/kg, significantly inhibits LPS-induced TNF production (45%) in comparison with the vehicle-treated group. PS166276, a 6-fold more potent compound compared with PS200981 in the cellular assay, was also tested in the same model. Figure 6B demonstrates that PS166276 is more efficacious than PS200981 in vivo. PS166276, at 30 mg/kg, inhibits 81% of LPS-induced TNF production versus the vehicle-treated group. No obvious toxicity was observed in PS200981- or PS166276-treated animals.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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sequestrants or inhibit the action of interleukin-1 have demonstrated efficacy as therapeutics in the treatment of chronic inflammatory diseases, e.g. rheumatoid arthritis and Crohn's disease. Although efficacious, the cost and route of administration of these biologicals are disadvantageous to patients and health care systems compared with a small-molecule p38 inhibitor that can be administered orally if one can be identified and advanced. We report here the identification of a triaminotriazine aniline amide series of potent and selective p38 inhibitors from a collection of large combinatorial libraries. Forty-one combinatorial libraries averaging 50,000 members per library were screened. An active triaminotriazine library was identified. A strong preference for the aniline amide was observed from the screening data. This library has been screened at 25 other kinases, and although subsets of this library have produced inhibitors of other kinases, the observed preference for the methylanilino amide is unique to p38 kinase. Other related substituents such as 3-amino benzamide and 2-amino-4-methyl benzamide were contained in the library but not found among the p38 inhibitors.
With one exception, all known p38 inhibitors bind to the ATP-binding pocket and inhibit the kinase by directly competing with the binding of ATP. Uniquely, the p38 inhibitor, BIRB-796, binds to a kinase specificity pocket and to the ATP-binding site. This dual binding mode results in a conformational change in p38 and slow association kinetics of binding (Pargellis et al., 2002). Kinetics studies have indicated that the current class of p38 inhibitors, like most other p38 inhibitors, competes with the binding of ATP.
Over their entire kinase domains, p38 and p38 have 83% sequence identity. Within the ATP-binding site, the two isoforms are nearly identical. P38 and are identical within the main hydrophobic pocket, including the key gate keeper residue for which they both have a Thr (Thr106 of p38). It is noteworthy that p38, p38, and most of the JNK and ERK kinases have a Met at the gatekeeper position. The key gatekeeper residue has been shown to be a critical determinant in kinase selectivity. This may explain the observation that all known p38 inhibitors, including our new series of p38 inhibitors, have limited selectivity between p38 and p38, while showing good selectivity against p38 and other protein kinases.
Mice null for the p38 allele die during early embryonic development. The development arrest suggests that the different p38 isozymes do not perform redundant activities, at least during embryonic development. p38-/- embryonic stem cells fail to activate Mapkap K2 in response to chemical stress inducers and generate minimal levels of interleukin-6 in response to interleukin-1, despite the fact that p38-/- embryonic stem cells express three other p38 kinases (Allen et al., 2000). These results, together with pharmacological studies using specific p38 inhibitors (Adams et al., 2001), indicate that p38 is the key p38 kinase involved in the inflammatory response. Evidence from p38 inhibitors that have limited selectivity against p38 and show no obvious adverse effects in animal tests and clinical trials suggests that some activity against p38 is also tolerated (Kumar et al., 2003).
PS200981, a p38 inhibitor identified from high-throughput screening, shows inhibition of p38 kinase and p38-mediated cellular assays (Fig. 3). This compound, and a highly related more potent analog PS166276, exhibit >25-fold selectivity against a panel of non-p38 kinases (Table 1). Furthermore, PS200981 and PS166276 were tested in vivo and inhibited 50 to 80% of the LPS-induced TNF production in mice (Fig. 6, A and B). These results indicate that the initial p38 hits, exemplified by PS200981, as well as early analogs such as PS166276, have suitable characteristics for further optimization and study.
PS166276, a highly related analog of PS200981, was used to further assess selectivity by analyzing cellular events. PS166276 did not inhibit several distinct signal transduction pathways, which includes the signaling cascades that lead to phosphorylation of ERK1/2, ATF-2, Stat5, and degradation of IB. Each of these signaling cascades consists of multiple kinases and other signaling proteins that lead to the components that are analyzed by Western blotting (Fig. 5). A total of at least 20 protein kinases are involved in these signaling pathways. Furthermore, PS166276 was not cytotoxic up to 100 µM, indicating that the actions of PS166276 are not due to toxicity or pleiotropic effects.
In contrast to PS200981 and PS166276, members of the current triaminotriazine aniline amides, members of the original series of triaryl-imidazoles such as SB203580, inhibit JNK2 (data not shown) and have been shown to be inhibitors of a number of other kinases including JNK3, CSNK1E, GAK, RICK, and CK1 (Godl et al., 2003; Fabian et al., 2005). It has also been observed that several known p38 inhibitors are more potent inhibitors of TNF expression in human peripheral blood mononuclear cells than would otherwise be expected given their p38 kinase IC50 values (Diller et al., 2005). This includes members of the triaryl-imidazole class of p38 inhibitors such as RWJ68354. Thus, there is reason to believe that the observed inhibition of TNF expression is due to the inhibition of something other than their activity at p38. In contrast, the good correspondence between kinase, cellular, and in vivo data observed for the current compounds is consistent with limited off-target effects and is probably due to the strong preference for the methylanilino amide synthon in this series.
In conclusion, we have identified a novel class of p38 kinase inhibitors that block in vitro p38 kinase, p38 cell-based activity, in vivo activity, and which as exemplars of screening hits (PS200981) and early analogs (PS166276) are highly selective against other kinases. Moreover, p38 kinase shows a strong synthon preference for the methylanilino amide at R2. This preferred methylanilino amide is a key component in the structure activity of these compounds and is deemed critical for the selectivity profile observed to date versus other kinases.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: MAP, mitogen-activated protein; TNF, tumor necrosis factor; MKK, MAP kinase kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; GST, glutathione S-transferase; IKK, IB kinase complex; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; PS200981, 3-(4-(1,4-diazepan-1-yl)-6-(((1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]heptan-2-yl)methylamino)-1,3,5-triazin-2-ylamino)-4-methylbenzamide; PS166276, (R)-3-(4-(isobutyl(methyl)amino)-6-(pyrrolidin-3-ylamino)-1,3,5-triazin-2-ylamino)-4-methylbenzamide; PBS, phosphate-buffered saline; VX-745, 5-(2,6-dichlorophenyl)-2-(phenylthio)-6H-pyrimido[1,6-b]pyridazin-6-one; BIRB-796, N-[3-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazol-5-yl]-N'-[4-[2-(4-morpholinyl)ethoxy]-1-naphthalenyl]-urea; SB203580, 4-[5-(4-fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-1H-imidazol-4-yl]-pyridine; RWJ67657, 4-(4-fluorophenyl)-2-(4-hydroxy-1-butynyl)-1-(3-phenylpropyl)-5-(4-pyridyl)imidazole "imidazopyridine"; RPR200765A, 4-[[trans-2-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-5-methyl-1,3-dioxan-5-yl]carbonyl]-morpholine; TPA, O-tetradecanoyl-phorbol 13-acetate.
Address correspondence to: Maria L. Webb, Pharmacopeia Drug Discovery Inc., P.O. Box 5350, Princeton, NJ 08543-5350. E-mail: Mwebb{at}pcop.com
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Km [ATP]. IC50 values are in micromolars.
