ABSTRACT
Targeted inhibitors of bromodomain and extraterminal (BET)-bromodomains and phosphatidylinositol-3-kinase (PI3K) signaling demonstrate potent but self-limited antilymphoma activity as single agents in the context of cellular Myelocytomatosis (cMYC) oncogene-dysregulation. However, combined PI3K and BET inhibition imparts synergistic anticancer activity with the potential for more sustained disease responses due to the mutual antagonism of compensatory epigenetic and signaling networks. Here, we describe the mechanistic and therapeutic validation of rationally designed dual PI3K/BET bromodomain inhibitors, built by linkage of established PI3K and BET inhibitor pharmacophores. The lead candidate demonstrates high selectivity, nanomolar range cellular potency, and compelling in vivo efficacy, including curative responses in the aggressive Eµ-Myc lymphoma model. These studies further support the therapeutic strategy of combined PI3K and BET inhibition and provide a potential step-change in approach to orthogonal MYC antagonism using optimized chimeric small-molecule technology.
Subject(s)
Lymphoma , Phosphatidylinositol 3-Kinases , Humans , Phosphatidylinositol 3-Kinase , Aggression , Epigenomics , Lymphoma/drug therapy , Phosphoinositide-3 Kinase InhibitorsABSTRACT
A novel series of TGX-221 analogues was prepared that include isosteric replacement of the 4H-pyrido[1,2-a]pyrimidin-4-one with a 4H-benzo[e][1,3]oxazin-4-one scaffold. The compounds that included an CH(CH3)NH type linker showed comparable activity to TGX-221 analogues with the isosterism supported by the comparative SAR analysis. The analogues containing an CH(CH3)O linker were less active but still showed useful SAR including a favoured o-methyl substitution.
Subject(s)
Morpholines , Pyrimidinones , Pyrimidinones/pharmacology , Structure-Activity RelationshipABSTRACT
To continue our study of 2-morpholino-benzoxazine based compounds, which show useful activity against PI3K family enzymes or antiplatelet activity, we designed and synthesized a series of linear 6.7-fused, 5,6-angular fused and 7,8-angular fused-aryl-morpholino-naphth-oxazines. The compounds were prepared from substituted 2-hydroxynaphthoic acid to give the corresponding thioxo analogues 8, 9, 15 and 19. The thioxo products were then converted to the morpholino substituted analogue. The aryl group was introduced by Suzuki coupling of bromo precursors. The products were evaluated for activity at PI3K family enzymes and as platelet aggregation inhibitors and compared to reported unsubstituted analogues. The linear 6.7-fused product 13a and 13b were moderated potent but selective PI3Kδ isoform inhibitors (IC50=7.7 and 5.61µM). Good antiplatelet activity was noticed for the angular 7,8-fused compounds 22a, b, k and l with IC50=3.0,14.0, 2.0 and 5.0µM respectively. The antiplatelet activity is independent of PDE3.
Subject(s)
Benzoxazines/pharmacology , DNA-Activated Protein Kinase/antagonists & inhibitors , Morpholinos/pharmacology , Phosphodiesterase 3 Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Platelet Aggregation Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacology , Benzoxazines/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , DNA-Activated Protein Kinase/metabolism , Humans , Morpholinos/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Phosphodiesterase 3 Inhibitors/chemistry , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/chemistry , Protein Kinase Inhibitors/chemistryABSTRACT
A series of PI3Kδ inhibitors derived from the pan-PI3K inhibitor ZSTK474 was prepared that target a non-conserved region of the catalytic site. Dependent upon the substituents present, these analogues show different levels of isoform selectivity and sensitivity to the mutation N836D in PI3Kδ. As a marker of 'on-target' activity and permeability, a selection of the most potent PI3Kδ inhibitors were shown to inhibit pAkt production in the Nawalma Burkitt lymphoma cell line.
Subject(s)
Enzyme Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Cell Line, Tumor , Humans , Isoenzymes/chemistry , Phosphatidylinositol 3-Kinases/chemistryABSTRACT
We report on 6 patients in our care who were harboring atherosclerotic plaque in the carotid arteries. This condition poses a risk of acute ischemic stroke and indicates potential atherosclerosis elsewhere in the vascular system. The plaque was revealed by routine ultrasound measurement of carotid intima-medial thickness (CIMT) defined as the distance between the lumen-intima interface and the media-adventitia interface. Recent improvements in image resolution and edge detection algorithms have resulted in improved reliability and clinical usefulness of the technology. The patients were enrolled in a systems-based functional medicine program of cardiology prevention to address root causes. The program provided personalized interventions that included drug therapy, dietary supplements, and lifestyle modification. The 6 patients followed the integrative regimen, which successfully managed existing cardiovascular symptoms and risk factors while keeping various biomarkers under control. However, they continued to exhibit carotid plaque with no improvement. A novel dietary supplement that targets endothelial glycocalyx regeneration was added to the personalized intervention programs. The supplement contains a proprietary extract of rhamnan sulfate from the green seaweed Monostroma nitidum. The 6 participants consumed the supplement daily, and their plaque burden was measured after 6 months using the same CIMT technology. In every case, the total plaque burden was reduced, with an average reduction in the 6 patients of 5.55 mm, which is statistically significant. Significant reductions in maximum carotid plaque thickness were also observed at the end of the 6 months. The study suggests that rhamnan sulfate from Monostroma nitidum may provide a safe and effective intervention for reducing atherosclerotic plaque, and should be evaluated as an adjunct therapy for prevention and treatment of cardiovascular disease.
ABSTRACT
Phosphoinositide 3-kinases (PI3K) hold significant therapeutic potential as novel targets for the treatment of cancer. ZSTK474 (4a) is a potent, pan-PI3K inhibitor currently under clinical evaluation for the treatment of cancer. Structural studies have shown that derivatisation at the 5- or 6-position of the benzimidazole ring may influence potency and isoform selectivity. However, synthesis of these derivatives by the traditional route results in a mixture of the two regioisomers. We have developed a straightforward regioselective synthesis that gave convenient access to 5- and 6-methoxysubstituted benzimidazole derivatives of ZSTK474. While 5-methoxy substitution abolished activity at all isoforms, the 6-methoxy substitution is consistently 10-fold more potent. This synthesis will allow convenient access to further 6-position derivatives, thus allowing the full scope of the structure-activity relationships of ZSTK474 to be probed.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Phosphoinositide-3 Kinase Inhibitors , Triazines/chemical synthesis , Aniline Compounds/chemistry , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Nitro Compounds/chemistry , Stereoisomerism , Structure-Activity Relationship , Triazines/chemistry , Triazines/pharmacologyABSTRACT
The binding mechanism of a new class of lipid-competitive, ATP non-competitive, p110α isoform-selective PI3K (phosphoinositide 3-kinase) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of non-conserved amino acids in the p110α and p110ß isoforms, we have identified three unique binding mechanisms for the p110α-selective inhibitors PIK-75, A-66S and J-32. Each of the inhibitor's p110α-isoform-selective binding was found to be due to interactions with different amino acids within p110. The PIK-75 interaction bound the non-conserved region 2 amino acid p110α Ser(773), A-66S bound the region 1 non-conserved amino acid p110α Gln(859), and J-32 binding had an indirect interaction with Lys(776) and Ile(771). The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.
Subject(s)
Amino Acids/metabolism , Mutagenesis, Site-Directed , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proline/analogs & derivatives , Thiazoles/metabolism , Amino Acids/genetics , Class I Phosphatidylinositol 3-Kinases , Class II Phosphatidylinositol 3-Kinases/genetics , Class II Phosphatidylinositol 3-Kinases/metabolism , Conserved Sequence/genetics , Dose-Response Relationship, Drug , Hydrazones/metabolism , Hydrazones/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Phosphatidylinositol 3-Kinases/genetics , Proline/genetics , Proline/metabolism , Protein Binding/genetics , Sulfonamides/metabolism , Sulfonamides/pharmacologyABSTRACT
The thiazolidinedione, compound 1, has previously shown pan-inhibition of the phosphoinositide 3-kinase (PI3K) class I isoforms. We hypothesized the derivatization of the thiazolidinedione core of compound 1 could introduce isoform selectivity. We report the synthesis, characterization, and inhibitory activity of a novel series of 4-iminothiazolidin-2-ones for inhibition of the class I PI3K isoforms. Their synthesis was successfully achieved by multiple pathways described in this paper. Initial in vitro data of 28 analogues demonstrated poor inhibition of all class I PI3K isoforms. However, we identified an alternate target, the phosphodiesterases, and present preliminary screening results showing improved inhibitory activity.
ABSTRACT
The combination of molecular modeling and X-ray crystallography has failed to yield a consensus model of the mechanism for selective binding of inhibitors to the phosphoinositide 3-kinase (PI3K) p110 α-isoform. Here we have used kinetic analysis to determine that the p110α-selective inhibitor 2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-α]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid (PIK-75) is a competitive inhibitor with respect to a substrate, phosphatidylinositol (PI) in contrast to most other PI3K inhibitors, which bind at or near the ATP site. Using sequence analysis and the existing crystal structures of inhibitor complexes with the p110γ and -δ isoforms, we have identified a new region of nonconserved amino acids (region 2) that was postulated to be involved in PIK-75 p110α selectivity. Analysis of region 2, using in vitro mutation of identified nonconserved amino acids to alanine, showed that Ser773 was a critical amino acid involved in PIK-75 binding, with an 8-fold-increase in the IC(50) compared with wild-type. Kinetic analysis showed that, with respect to PI, the PIK-75 K(i) for the isoform mutant S773D increased 64-fold compared with wild-type enzyme. In addition, a nonconserved amino acid, His855, from the previously identified region 1 of nonconserved amino acids, was found to be involved in PIK-75 binding. These results show that these two regions of nonconserved amino acids that are close to the substrate binding site could be targeted to produce p110α isoform-selective inhibitors.
Subject(s)
Amino Acids/chemistry , Hydrazones/pharmacology , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Sulfonamides/pharmacology , Amino Acid Sequence , Amino Acids/genetics , Amino Acids/metabolism , Animals , Binding Sites/physiology , Cell Line , Class I Phosphatidylinositol 3-Kinases , Conserved Sequence , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Hydrazones/metabolism , Insecta , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/metabolism , Sulfonamides/metabolismABSTRACT
PDE4 inhibitors have been identified as therapeutic targets for a variety of conditions, particularly inflammatory diseases. We have serendipitously identified a novel class of phosphodiesterase 4 (PDE4) inhibitor during a study to discover antagonists of the parathyroid hormone receptor. X-ray crystallographic studies of PDE4D2 complexed to four potent inhibitors reveal the atomic details of how they inhibit the enzyme and a notable contrast to another recently reported thiophene-based inhibitor.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 4/chemistry , Models, Molecular , Phosphodiesterase 4 Inhibitors/chemistry , Thiophenes/chemical synthesis , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Phosphodiesterase 4 Inhibitors/chemical synthesis , Protein Binding , Thiophenes/chemistry , Thiophenes/pharmacologyABSTRACT
Arterial thrombosis causes heart attacks and most strokes and is the most common cause of death in the world. Platelets are the cells that form arterial thrombi, and antiplatelet drugs are the mainstay of heart attack and stroke prevention. Yet, current drugs have limited efficacy, preventing fewer than 25% of lethal cardiovascular events without clinically relevant effects on bleeding. The key limitation on the ability of all current drugs to impair thrombosis without causing bleeding is that they block global platelet activation, thereby indiscriminately preventing platelet function in hemostasis and thrombosis. Here, we identify an approach with the potential to overcome this limitation by preventing platelet function independently of canonical platelet activation and in a manner that appears specifically relevant in the setting of thrombosis. Genetic or pharmacological targeting of the class II phosphoinositide 3-kinase (PI3KC2α) dilates the internal membrane reserve of platelets but does not affect activation-dependent platelet function in standard tests. Despite this, inhibition of PI3KC2α is potently antithrombotic in human blood ex vivo and mice in vivo and does not affect hemostasis. Mechanistic studies reveal this antithrombotic effect to be the result of impaired platelet adhesion driven by pronounced hemodynamic shear stress gradients. These findings demonstrate an important role for PI3KC2α in regulating platelet structure and function via a membrane-dependent mechanism and suggest that drugs targeting the platelet internal membrane may be a suitable approach for antithrombotic therapies with an improved therapeutic window.
Subject(s)
Blood Platelets , Thrombosis , Animals , Hemostasis , Mice , Phosphatidylinositol 3-Kinases , Platelet Activation , Platelet Aggregation , Thrombosis/drug therapyABSTRACT
The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr(886) and Lys(890) of the PI3Kgamma isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the beta isoform (Glu(858) and Asp(862)) and those in the alpha isoform (His(855) and Gln(859)) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of beta-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the alpha-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kbeta activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the alpha-isoform-selectivity of the compounds studied.
Subject(s)
Enzyme Inhibitors/chemistry , Phosphatidylinositol 3-Kinases/chemistry , Phosphoinositide-3 Kinase Inhibitors , Binding Sites , Catalysis , Catalytic Domain , Enzyme Inhibitors/pharmacology , Kinetics , Mutagenesis, Site-Directed , Phosphatidylinositol 3-Kinases/genetics , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
AMP-activated protein kinase (AMPK) is a multisubstrate enzyme activated by increases in AMP during metabolic stress caused by exercise, hypoxia, lack of cell nutrients, as well as hormones, including adiponectin and leptin. Furthermore, metformin and rosiglitazone, frontline drugs used for the treatment of type II diabetes, activate AMPK. Mammalian AMPK is an alphabetagamma heterotrimer with multiple isoforms of each subunit comprising alpha1, alpha2, beta1, beta2, gamma1, gamma2, and gamma3, which have varying tissue and subcellular expression. Mutations in the AMPK gamma subunit cause glycogen storage disease in humans, but the molecular relationship between glycogen and the AMPK/Snf1p kinase subfamily has not been apparent. We show that the AMPK beta subunit contains a functional glycogen binding domain (beta-GBD) that is most closely related to isoamylase domains found in glycogen and starch branching enzymes. Mutation of key glycogen binding residues, predicted by molecular modeling, completely abolished beta-GBD binding to glycogen. AMPK binds to glycogen but retains full activity. Overexpressed AMPK beta1 localized to specific mammalian subcellular structures that corresponded with the expression pattern of glycogen phosphorylase. Glycogen binding provides an architectural link between AMPK and a major cellular energy store and juxtaposes AMPK to glycogen bound phosphatases.
Subject(s)
Glycogen/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Stress, Physiological/metabolism , AMP-Activated Protein Kinases , Amino Acid Sequence , Animals , Gene Expression , Glycogen/pharmacology , Glycogen Phosphorylase/chemistry , Glycogen Phosphorylase/genetics , Glycogen Phosphorylase/metabolism , Glycogen Synthase/metabolism , Models, Molecular , Molecular Sequence Data , Multienzyme Complexes/genetics , Multienzyme Complexes/ultrastructure , Phylogeny , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/ultrastructure , Protein Structure, Tertiary , Protein Subunits , Rats , Sequence Homology, Amino AcidABSTRACT
The synthesis of 6-aryl, 8- aryl, and 8-aryl-6-chloro-2-morpholino-1,3-benzoxazines with potent activity against PI3K and DNA-PK is described. Synthesis of thirty one analogues was facilitated by an improved synthesis of 3-bromo-2-hydroxybenzoic acid 13 by de-sulphonation of 3-bromo-2-hydroxy-5-sulfobenzoic acid 12 en route to 2-methylthio-substituted-benzoxazine intermediates 17-19. From this series, compound 20k (LTURM34) (dibenzo[b,d]thiophen-4-yl) (IC50 = 0.034 µM) was identified as a specific DNA-PK inhibitor, 170 fold more selective for DNA-PK activity compared to PI3K activity. Other compounds of the series show markedly altered selectivity for various PI3K isoforms including compound 20i (8-(naphthalen-1-yl) a potent and quite selective PI3Kδ inhibitor (IC50 = 0.64 µM). Finally, nine compounds were evaluated and showed antiproliferative activity against an NCI panel of cancer cell lines. Compound 20i (8-(naphthalen-1-yl) showed strong anti-proliferative activity against A498 renal cancer cells that warrants further investigation.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Benzoxazines/chemistry , Benzoxazines/pharmacology , DNA-Activated Protein Kinase/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Antineoplastic Agents/chemical synthesis , Benzoxazines/chemical synthesis , Cell Line, Tumor , Cell Proliferation/drug effects , DNA-Activated Protein Kinase/metabolism , Humans , Naphthalenes/chemical synthesis , Naphthalenes/chemistry , Naphthalenes/pharmacology , Neoplasms/drug therapy , Neoplasms/enzymology , Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Structure-Activity Relationship , Thiophenes/chemical synthesis , Thiophenes/chemistry , Thiophenes/pharmacologyABSTRACT
A series of amino-substituted triazines were developed and examined for PI3Kß inhibition and anti-platelet function. Structural adaptations of a morpholine ring of the prototype pan-PI3K inhibitor ZSTK474 yielded PI3Kß selective compounds, where the selectivity largely derives from an interaction with the non-conserved Asp862 residue, as shown by site directed mutagenesis. The most PI3Kß selective inhibitor from the series was studied in detail through a series of in vitro and in vivo functional studies. MIPS-9922, 10 potently inhibited ADP-induced washed platelet aggregation. It also inhibited integrin αIIbß3 activation and αIIbß3 dependent platelet adhesion to immobilized vWF under high shear. It prevented arterial thrombus formation in the in vivo electrolytic mouse model of thrombosis without inducing prolonged bleeding or excess blood loss.
Subject(s)
Drug Discovery , Enzyme Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Platelet Aggregation Inhibitors/pharmacology , Triazines/pharmacology , Animals , Enzyme Inhibitors/pharmacokinetics , Humans , Male , Mice , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/pharmacokinetics , Rats , Triazines/pharmacokineticsABSTRACT
The Class II PI3 kinases are emerging from the shadows of their Class I cousins. The data emerging from PIK3C2 genetic modification studies and from siRNA knockdown suggest important roles in physiology and pathology. With some well-studied Class I isoform inhibitors showing strong Class II activity and a wealth of crystallographic information available, the structural similarity of these isoforms to Class I provides both the opportunity and the challenge in design of selective pharmacological inhibitors.
ABSTRACT
Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, the causative agents of severe malaria and toxoplasmosis, respectively, undergo several critical developmental transitions during their lifecycle. Most important for human pathogenesis is the asexual cycle, in which parasites undergo rounds of host cell invasion, replication, and egress (exit), destroying host cell tissue in the process. Previous work has identified important roles for Protein Kinase G (PKG) and Protein Kinase A (PKA) in parasite egress and invasion, yet little is understood about the regulation of cyclic nucleotides, cGMP and cAMP, that activate these enzymes. To address this, we have focused upon the development of inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) to block the breakdown of cyclic nucleotides. This was done by repurposing human PDE inhibitors noting various similarities of the human and apicomplexan PDE binding sites. The most potent inhibitors blocked the in vitro proliferation of P. falciparum and T. gondii more potently than the benchmark compound zaprinast. 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO) was found to be a potent inhibitor of recombinant P. falciparum PfPDEα and activated PKG-dependent egress of T. gondii and P. falciparum, likely by promoting the exocytosis of micronemes, an activity that was reversed by a specific Protein Kinase G inhibitor. BIPPO also promotes cAMP-dependent phosphorylation of a P. falciparum ligand critical for host cell invasion, suggesting that the compound inhibits single or multiple PDE isoforms that regulate both cGMP and cAMP levels. BIPPO is therefore a useful tool for the dissection of signal transduction pathways in apicomplexan parasites.
Subject(s)
Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/pharmacology , Plasmodium falciparum/drug effects , Toxoplasma/drug effects , Antiprotozoal Agents/chemical synthesis , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/pharmacology , Chemistry Techniques, Synthetic , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Drug Evaluation, Preclinical/methods , Female , Humans , Phosphodiesterase Inhibitors/chemical synthesis , Phosphorylation/drug effects , Plasmodium falciparum/physiology , Purinones/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Sequence Alignment , Sequence Homology, Amino Acid , Structural Homology, Protein , Toxoplasma/enzymology , Toxoplasma/physiologyABSTRACT
Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.
Subject(s)
Phenylalanine Hydroxylase/chemistry , Phenylalanine Hydroxylase/physiology , Amino Acid Sequence , Animals , Binding Sites , Catalytic Domain , Cyclic AMP-Dependent Protein Kinases/metabolism , Escherichia coli/enzymology , Gene Expression Regulation , Insecta/enzymology , Models, Biological , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular/methods , Peptides/chemical synthesis , Phenylalanine/metabolism , Phenylalanine/pharmacology , Phenylalanine Hydroxylase/genetics , Phenylalanine Hydroxylase/metabolism , Phosphorylation , Rats , Spectrum AnalysisABSTRACT
We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP2, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP2 molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.
Subject(s)
Phosphatidylinositol 3-Kinases/chemistry , Amino Acid Sequence , Animals , Binding Sites , Boron Compounds/chemistry , Models, Molecular , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Protein Conformation , Sf9 Cells , Signal Transduction , Spodoptera , src Homology DomainsABSTRACT
The p110ß isoform of PI3 kinase (PI3Kß) has been implicated in pathological disorders such as thrombosis and cancer and a number of PI3Kß-selective inhibitors have recently progressed into clinical studies. Although crystallography studies identify a binding site conformation favored by the inhibitors, no specific interaction explains the observed selectivity. Using site-directed mutagenesis we have identified a specific tyrosine residue of the binding site Y778 that dictates the ability of the PI3Kß isoform to bind these inhibitors. When mutated to isoleucine, PI3Kß has reduced ability to present a specific cryptic binding site into which a range of reported PI3Kß inhibitors can bind, and conversely when tyrosine is introduced into the same position in PI3Kα, the same inhibitors gain potency. The results provide a cogent explanation for the selectivity profiles displayed by these PI3K inhibitors and maybe others as well.