The Effect of a Dietary Supplement Containing Rhamnan Sulfate from Monostroma nitidum on Carotid Atherosclerotic Plaque: A Case Series.

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.

Rationally designed chimeric PI3K-BET bromodomain inhibitors elicit curative responses in MYC-driven lymphoma.

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.

Synthesis and biological evaluation of 4H-benzo[e][1,3]oxazin-4-ones analogues of TGX-221 as inhibitors of PI3Kß.

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.

Disrupting the platelet internal membrane via PI3KC2α inhibition impairs thrombosis independently of canonical platelet activation.

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.

Synthesis of linear and angular aryl-morpholino-naphth-oxazines, their DNA-PK, PI3K, PDE3A and antiplatelet activity.

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.

Development of single and mixed isoform selectivity PI3Kδ inhibitors by targeting Asn836 of PI3Kδ.

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.

Discovery and antiplatelet activity of a selective PI3Kß inhibitor (MIPS-9922).

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.

Synthesis, structure elucidation, DNA-PK and PI3K and anti-cancer activity of 8- and 6-aryl-substituted-1-3-benzoxazines.

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.

Identification of potent phosphodiesterase inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites.

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.

Class II but Not Second Class-Prospects for the Development of Class II PI3K Inhibitors.

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.

Structural basis of nSH2 regulation and lipid binding in PI3Kα.

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.

L-Aminoacyl-triazine derivatives are isoform-selective PI3Kß inhibitors that target non-conserved Asp862 of PI3Kß

A series of aminoacyl-triazine derivatives based upon the pan-PI3K inhibitor ZSTK474 were identified as potent and isoform selective inhibitors of PI3Kß. The compounds showed selectivity based upon stereochemistry with L-amino acyl derivatives preferring PI3Kß while their D-congeners favoured PI3Kδ. The mechanistic basis of this inhibition was studied using site-directed mutants. One Asp residue, D862 was identified as a critical participant in binding to the PI3Kß-selective inhibitors distinguishing this class from other reported PI3Kß-selective inhibitors. The compounds show strong inhibition of cellular Akt phosphorylation and growth of PTEN-deficient MD-MBA-468 cells.

Potent inhibitors of phosphatidylinositol 3 (PI3) kinase that have antiproliferative activity only when delivered as prodrug forms.

Prodrugs for PI3K: A series of substituted analogues of the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002 were prepared and found to potently inhibit the isolated enzyme but not MCF7 cell proliferation. Two tetrazolyl-substituted analogues were further derivatized as prodrugs resulting in restoration of cell-based activity. These data provide a conceptual model for development of tumor-targeting prodrug forms of cell-impermeable PI3K inhibitors.

Mechanisms of PI3Kß-selective inhibition revealed by reciprocal mutagenesis.

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.

Regioselective synthesis of 5- and 6-methoxybenzimidazole-1,3,5-triazines as inhibitors of phosphoinositide 3-kinase.

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.

Definition of the binding mode of a new class of phosphoinositide 3-kinase α-selective inhibitors using in vitro mutagenesis of non-conserved amino acids and kinetic analysis.

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.

Synthesis and Pharmacological Evaluation of 4-Iminothiazolidinones for Inhibition of PI3 Kinase.

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.

Thiophene inhibitors of PDE4: crystal structures show a second binding mode at the catalytic domain of PDE4D2.

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.

Isoform-selective inhibition of phosphoinositide 3-kinase: identification of a new region of nonconserved amino acids critical for p110α inhibition.

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.

Thiazolidinedione-based PI3Kα inhibitors: an analysis of biochemical and virtual screening methods.

A series of synthesized and commercially available compounds were assessed against PI3Kα for in vitro inhibitory activity and the results compared to binding calculated in silico. Using published crystal structures of PI3Kγ and PI3Kδ co-crystallized with inhibitors as a template, docking was able to identify the majority of potent inhibitors from a decoy set of 1000 compounds. On the other hand, PI3Kα in the apo-form, modeled by induced fit docking, or built as a homology model gave only poor results. A PI3Kα homology model derived from a ligand-bound PI3Kδ crystal structure was developed that has a good ability to identify active compounds. The docking results identified binding poses for active compounds that differ from those identified to date and can contribute to our understanding of structure-activity relationships for PI3K inhibitors.