PDK1:PKCα heterodimer association-dissociation dynamics in single-molecule diffusion tracks on a target membrane.

Previous studies have documented the formation of a heterodimer between the two protein kinases PDK1 and PKCα on a lipid bilayer containing their target lipids. This work investigates the association-dissociation kinetics of this PDK1:PKCα heterodimer. The approach monitors the two-dimensional diffusion of single, membrane-associated PDK1 molecules for diffusivity changes as PKCα molecules bind and unbind. In the absence of PKCα, a membrane-associated PDK1 molecule exhibits high diffusivity (or large diffusion constant, D) because its membrane-contacting PH domain binds the target PIP3 lipid headgroup with little bilayer penetration, yielding minimal frictional drag against the bilayer. In contrast, membrane-associated PKCα contacts the bilayer via its C1A, C1B, and C2 domains, which each bind at least one target lipid with significant bilayer insertion, yielding a large frictional drag and low diffusivity. The present findings reveal that individual fluor-PDK1 molecules freely diffusing on the membrane surface undergo reversible switching between distinct high and low diffusivity states, corresponding to the PDK1 monomer and the PDK1:PKCα heterodimer, respectively. The observed single-molecule diffusion trajectories are converted to step length time courses, then subjected to two-state, hidden Markov modeling and dwell time analysis. The findings reveal that both the PDK1 monomer state and the PDK1:PKCα heterodimer state decay via simple exponential kinetics, yielding estimates of rate constants for state switching in both directions. Notably, the PDK1:PKCα heterodimer has been shown to competitively inhibit PDK1 phosphoactivation of AKT1, and is believed to play a tumor suppressor role by limiting excess activation of the highly oncogenic PDK1/AKT1/mTOR pathway. Thus, the present elucidation of the PDK1:PKCα association-dissociation kinetics has important biological and medical implications. More broadly, the findings illustrate the power of single-molecule diffusion measurements to reveal the kinetics of association-dissociation events in membrane signaling reactions that yield a large change in diffusive mobility.

Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation.

The eukaryotic kinase domain has multiple intrinsically disordered regions whose conformation dictates kinase activity. Small molecule kinase inhibitors (SMKIs) rely on disrupting the active conformations of these disordered regions to inactivate the kinase. While SMKIs are selected for their ability to cause this disruption, the allosteric effects of conformational changes in disordered regions is limited by a lack of dynamic information provided by traditional structural techniques. In this study, we integrated multiscale molecular dynamics simulations with FRET sensors to characterize a novel allosteric mechanism that is selectively triggered by SMKI binding to the protein kinase Cα domain. The indole maleimide inhibitors BimI and sotrastaurin were found to displace the Gly-rich loop (G-loop) that normally shields the ATP-binding site. Displacement of the G-loop interferes with a newly identified, structurally conserved binding pocket for the C1a domain on the N lobe of the kinase domain. This binding pocket, in conjunction with the N-terminal regulatory sequence, masks a diacylglycerol (DAG) binding site on the C1a domain. SMKI-mediated displacement of the G-loop released C1a and exposed the DAG binding site, enhancing protein kinase Cα translocation both to synthetic lipid bilayers and to live cell membranes in the presence of DAG. Inhibitor chemotype determined the extent of the observed allosteric effects on the kinase domain and correlated with the extent of membrane recruitment. Our findings demonstrate the allosteric effects of SMKIs beyond the confines of kinase catalytic conformation and provide an integrated computational-experimental paradigm to investigate parallel mechanisms in other kinases.

Identification, characterization, and comparison of n-alkanols and anesthetics binding to the C1b subdomain of protein kinase cα: similar function with different binding sites.

Protein kinase C (PKC) is a family of lipid-activated enzymes involved in anesthetic preconditioning signaling pathways. Previously, n-alkanols and general anesthetics have been found to activate PKC by binding to the kinase C1B subdomain. In the present study, we attempt to ascertain the molecular mechanism and interaction mode of human PKCα C1B subdomain with a variety of exogenous n-alkanols and volatile general anesthetics as well as endogenous activator phorbol ester (PE) and co-activator diacylglycerol (DG). Systematic bioinformatics analysis identifies three spatially vicinal sites on the subdomain surface to potentially accommodate small-molecule ligands, where the site 1 is a narrow, amphipathic pocket, the site 2 is a wide, flat and hydrophobic pocket, and the site 3 is a rugged, polar pocket. Further interaction modeling reveals that site 1 is the cognate binding region of natural PE activator, which can moderately simulate the kinase activity in an independent manner. The short-chain n-alkanols are speculated to also bind at the site to competitively inhibit PE-induced kinase activation. The long-chain n-alkanols and co-activator DG are found to target site 2 in a nonspecific manner, while the volatile anesthetics prefer to interact with site 3 in a specific manner. Since the site 1 is composed of two protein loops that are also shared by sites 2 and 3, binding of n-alkanols, DG and anesthetics to sites 2 and 3 can trigger a conformational displacement on the two loops, which enlarges the pocket size and changes the pocket configuration of site 1 through an allosteric mechanism, consequently enhancing kinase activation by improving PE affinity to the site.

Structures of the PKA RIα Holoenzyme with the FLHCC Driver J-PKAcα or Wild-Type PKAcα.

Fibrolamellar hepatocellular carcinoma (FLHCC) is driven by J-PKAcα, a kinase fusion chimera of the J domain of DnaJB1 with PKAcα, the catalytic subunit of protein kinase A (PKA). Here we report the crystal structures of the chimeric fusion RIα2:J-PKAcα2 holoenzyme formed by J-PKAcα and the PKA regulatory (R) subunit RIα, and the wild-type (WT) RIα2:PKAcα2 holoenzyme. The chimeric and WT RIα holoenzymes have quaternary structures different from the previously solved WT RIß and RIIß holoenzymes. The WT RIα holoenzyme showed the same configuration as the chimeric RIα2:J-PKAcα2 holoenzyme and a distinct second conformation. The J domains are positioned away from the symmetrical interface between the two RIα:J-PKAcα heterodimers in the chimeric fusion holoenzyme and are highly dynamic. The structural and dynamic features of these holoenzymes enhance our understanding of the fusion chimera protein J-PKAcα that drives FLHCC as well as the isoform specificity of PKA.

Structural Basis of Protein Kinase Cα Regulation by the C-Terminal Tail.

Protein kinase C (PKC) isoenzymes are multi-modular proteins activated at the membrane surface to regulate signal transduction processes. When activated by second messengers, PKC undergoes a drastic conformational and spatial transition from the inactive cytosolic state to the activated membrane-bound state. The complete structure of either state of PKC remains elusive. We demonstrate, using NMR spectroscopy, that the isolated Ca2+-sensing membrane-binding C2 domain of the conventional PKCα interacts with a conserved hydrophobic motif of the kinase C-terminal region, and we report a structural model of the complex. Our data suggest that the C-terminal region plays a dual role in regulating the PKC activity: activating, through sensitization of PKC to intracellular Ca2+ oscillations; and auto-inhibitory, through its interaction with a conserved positively charged region of the C2 domain.

A recurrent kinase domain mutation in PRKCA defines chordoid glioma of the third ventricle.

Chordoid glioma is a rare brain tumor thought to arise from specialized glial cells of the lamina terminalis along the anterior wall of the third ventricle. Despite being histologically low-grade, chordoid gliomas are often associated with poor outcome, as their stereotypic location in the third ventricle makes resection challenging and efficacious adjuvant therapies have not been developed. Here we performed genomic profiling on 13 chordoid gliomas and identified a recurrent D463H missense mutation in PRKCA in all tumors, which localizes in the kinase domain of the encoded protein kinase C alpha (PKCα). Expression of mutant PRKCA in immortalized human astrocytes led to increased phospho-ERK and anchorage-independent growth that could be blocked by MEK inhibition. These studies define PRKCA as a recurrently mutated oncogene in human cancer and identify a potential therapeutic vulnerability in this uncommon brain tumor.

Protein kinase C-α and the regulation of diverse cell responses.

Protein kinase C (PKC) comprises a family of lipid-sensitive enzymes that have been involved in a broad range of cellular functions. PKC-α is a member of classical PKC with ubiquitous expression and different cellular localization. This unique PKC isoform is activated by various signals which evoke lipid hydrolysis, after activation it interacts with various adapter proteins and is localized to specific cellular compartments where it is devised to work. The universal expression and activation by various stimuli make it a perfect player in uncountable cellular functions including differentiation, proliferation, apoptosis, cellular transformation, motility, adhesion and so on. However, these functions are not intrinsic properties of PKC-α, but depend on cell types and conditions. The activities of PKC-α are managed by the various pharmacological activators/inhibitors and antisense oligonucleotides. The aim of this review is to elaborate the structural feature, and provide an insight into the mechanism of PKC-α activation and regulation of its key biological functions in different cellular compartments to develop an effective pharmacological approach to regulate the PKC-α signal array.

Distinct structural mechanisms determine substrate affinity and kinase activity of protein kinase Cα.

Protein kinase Cα (PKCα) belongs to the family of AGC kinases that phosphorylate multiple peptide substrates. Although the consensus sequence motif has been identified and used to explain substrate specificity for PKCα, it does not inform the structural basis of substrate-binding and kinase activity for diverse substrates phosphorylated by this kinase. The transient, dynamic, and unstructured nature of this protein-protein interaction has limited structural mapping of kinase-substrate interfaces. Here, using multiscale MD simulation-based predictions and FRET sensor-based experiments, we investigated the conformational dynamics of the kinase-substrate interface. We found that the binding strength of the kinase-substrate interaction is primarily determined by long-range columbic interactions between basic (Arg/Lys) residues located N-terminally to the phosphorylated Ser/Thr residues in the substrate and by an acidic patch in the kinase catalytic domain. Kinase activity stemmed from conformational flexibility in the region C-terminal to the phosphorylated Ser/Thr residues. Flexibility of the substrate-kinase interaction enabled an Arg/Lys two to three amino acids C-terminal to the phosphorylated Ser/Thr to prime a catalytically active conformation, facilitating phosphoryl transfer to the substrate. The structural mechanisms determining substrate binding and catalytic activity formed the basis of diverse binding affinities and kinase activities of PKCα for 14 substrates with varying degrees of sequence conservation. Our findings provide insight into the dynamic properties of the kinase-substrate interaction that govern substrate binding and turnover. Moreover, this study establishes a modeling and experimental method to elucidate the structural dynamics underlying substrate selectivity among eukaryotic kinases.

Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1.

BACKGROUND: Resveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol's several biological targets is Ca2+-sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca2+-sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release. METHODS: To test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5'-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons. RESULTS: Resveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1H567K, a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket. CONCLUSIONS AND SIGNIFICANCE: This study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases.

Exploring the influence of indololactone structure on selectivity for binding to the C1 domains of PKCα, PKCε, and RasGRP.

C1 domain-containing proteins, such as protein kinase C (PKC), have a central role in cellular signal transduction. Their involvement in many diseases, including cancer, cardiovascular disease, and immunological and neurological disorders has been extensively demonstrated and has prompted a search for small molecules to modulate their activity. By employing a diacylglycerol (DAG)-lactone template, we have been able to develop ultra potent analogs of diacylglycerol with nanomolar binding affinities approaching those of complex natural products such as phorbol esters and bryostatins. One current challenge is the development of selective ligands capable of discriminating between different protein family members. Recently, structure-activity relationship studies have shown that the introduction of an indole ring as a DAG-lactone substituent yielded selective Ras guanine nucleotide-releasing protein (RasGRP1) activators when compared to PKCα and PKCε. In the present work, we examine the effects of ligand selectivity relative to the orientation of the indole ring and the nature of the DAG-lactone template itself. Our results show that the indole ring must be attached to the lactone moiety through the sn-2 position in order to achieve RasGRP1 selectivity.

Heterogeneous nuclear ribonucleoprotein M associates with mTORC2 and regulates muscle differentiation.

Mammalian target of rapamycin (mTOR) plays a range of crucial roles in cell survival, growth, proliferation, metabolism, and morphology. However, mTOR forms two distinct complexes, mTOR complex 1 and mTOR complex 2 (mTORC1 and mTORC2), via association with a series of different components; this allows the complexes to execute their wide range of functions. This study explores further the composition of the mTORC2 complex. Utilizing Rictor knock-out cells, immunoprecipitation and mass spectrometry, a novel Rictor associated protein, heterogeneous nuclear ribonucleoprotein M (hnRNP M), was identified. The association between hnRNP M and Rictor was verified using recombinant and endogenous protein and the binding site was found to be within aa 1~532 of hnRNP M. The presence of hnRNP M significantly affects phosphorylation of SGK1 S422, but not of Akt S473, PKCα S657 and PKCζ T560. Furthermore, hnRNP M also plays a critical role in muscle differentiation because knock-down of either hnRNP M or Rictor in C2C12 myoblasts reduced differentiation. This decrease is able to be rescued by overexpression SGK S422D in hnRNP M knockdown C2C12 myoblasts. Taken together, we have identified a novel Rictor/mTOR binding molecule, hnRNP M, that allows mTORC2 signaling to phosphorylate SGK1 thus regulating muscle differentiation.

The Role of Regulatory Domains in Maintaining Autoinhibition in the Multidomain Kinase PKCα.

Resolving the conformational dynamics of large multidomain proteins has proven to be a significant challenge. Here we use a variety of techniques to dissect the roles of individual protein kinase Cα (PKCα) regulatory domains in maintaining catalytic autoinhibition. We find that whereas the pseudosubstrate domain is necessary for autoinhibition it is not sufficient. Instead, each regulatory domain (C1a, C1b, and C2) appears to strengthen the pseudosubstrate-catalytic domain interaction in a nucleotide-dependent manner. The pseudosubstrate and C1a domains, however, are minimally essential for maintaining the inactivated state. Furthermore, disrupting known interactions between the C1a and other regulatory domains releases the autoinhibited interaction and increases basal activity. Modulating this interaction between the catalytic and regulatory domains reveals a direct correlation between autoinhibition and membrane translocation following PKC activation.

Investigation of the Effect of Bilayer Composition on PKCα-C2 Domain Docking Using Molecular Dynamics Simulations.

The protein kinase Cα (PKCα) enzyme is a member of a broad family of serine/threonine kinases, which are involved in varied cellular signaling pathways. The initial step of PKCα activation involves the C2 subunit docking with the cell membrane, which is followed by interactions of the C1 domains with diacylglycerol (DAG) in the membrane. Notably, the molecular mechanisms of these interactions remain poorly understood, especially what effects, if any, DAG may have on the initial C2 docking. To further understand this process, we have performed a series of conventional molecular dynamics simulations to systematically investigate the interaction between PKCα-C2 domains and lipid bilayers with different compositions to examine the effects of POPS, PIP2, and 1-palmitoyl-2-oleoyl-sn-glycerol (POG) on domain docking. Our results show that the PKCα-C2 domain does not interact with the bilayer surface in the absence of POPS and PIP2. In contrast, the inclusion of POPS and PIP2 to the bilayer resulted in strong domain docking in both perpendicular and parallel orientations, whereas the further inclusion of POG resulted in only parallel domain docking. In addition, lysine residues in the C2 domain formed hydrogen bonds with PIP2 molecule bilayers containing POG. These effects were further explored with umbrella sampling calculations to estimate the free energy of domain docking to the lipid bilayer in the presence of one or two PIP2 molecules. The results show that the binding of one or two PIP2 molecules is thermodynamically favorable, although stronger in bilayers lacking POG. However, in POG-containing bilayers, the binding mode of the C2 domain appears to be more flexible, which may have implications for activation of full-length PKCα. Together, our results shed new insights into the process of C2 bilayer binding and suggest new mechanisms for the roles of different phospholipids in the activation process of PKCα.

Curcumin Inhibits Protein Kinase Cα Activity by Binding to Its C1 Domain.

Curcumin is a polyphenolic nutraceutical that acts on multiple biological targets, including protein kinase C (PKC). PKC is a family of serine/threonine kinases central to intracellular signal transduction. We have recently shown that curcumin selectively inhibits PKCα, but not PKCε, in CHO-K1 cells [Pany, S. (2016) Biochemistry 55, 2135-2143]. To understand which domain(s) of PKCα is responsible for curcumin binding and inhibitory activity, we made several domain-swapped mutants in which the C1 (combination of C1A and C1B) and C2 domains are swapped between PKCα and PKCε. Phorbol ester-induced membrane translocation studies using confocal microscopy and immunoblotting revealed that curcumin inhibited phorbol ester-induced membrane translocation of PKCε mutants, in which the εC1 domain was replaced with αC1, but not the PKCα mutant in which αC1 was replaced with the εC1 domain, suggesting that αC1 is a determinant for curcumin's inhibitory effect. In addition, curcumin inhibited membrane translocation of PKCε mutants, in which the εC1A and εC1B domains were replaced with the αC1A and αC1B domains, respectively, indicating the role of both αC1A and αC1B domains in curcumin's inhibitory effects. Phorbol 13-acetate inhibited the binding of curcumin to αC1A and αC1B with IC50 values of 6.27 and 4.47 µM, respectively. Molecular docking and molecular dynamics studies also supported the higher affinity of curcumin for αC1B than for αC1A. The C2 domain-swapped mutants were inactive in phorbol ester-induced membrane translocation. These results indicate that curcumin binds to the C1 domain of PKCα and highlight the importance of this domain in achieving PKC isoform selectivity.

C2-domain mediated nano-cluster formation increases calcium signaling efficiency.

Conventional protein kinase Cs (cPKCs) are key signaling proteins for transducing intracellular Ca2+ signals into downstream phosphorylation events. However, the lifetime of individual membrane-bound activated cPKCs is an order of magnitude shorter than the average time needed for target-protein phosphorylation. Here, we employed intermolecular Förster resonance energy transfer (FRET) in living cells combined with computational analysis to study the spatial organization of cPKCs bound to the plasma membrane. We discovered Ca2+-dependent cPKC nano-clusters that significantly extend cPKC's plasma-membrane residence time. These protein patterns resulted from self-assembly mediated by Ca2+-binding C2-domains, which are widely used for membrane-targeting of Ca2+-sensing proteins. We also established clustering of other unrelated C2-domain containing proteins, suggesting that nano-cluster formation is a key step for efficient cellular Ca2+-signaling.

Dynamic Response of the C2 Domain of Protein Kinase Cα to Ca2+ Binding.

Ca2+-dependent conserved-region 2 (C2) domains target their host signaling proteins to anionic membranes. The Ca2+-binding event is a prerequisite for membrane association. Here, we investigate multiscale metal-ion-dependent dynamics of the C2 domain of protein kinase Cα (C2α) using NMR spectroscopy. Interactions with metal ions attenuate microsecond-timescale motions of the loop regions, indicating that preorganization of the metal-binding loops occurs before membrane insertion. Binding of a full complement of Ca2+ ions has a profound effect on the millisecond-timescale dynamics of the N- and C-terminal regions of C2α. We propose that Ca2+ binding allosterically destabilizes the terminal regions of C2α and thereby facilitates the conformational rearrangement necessary for full membrane insertion and activation of protein kinase Cα.

Calcium Stimulates Self-Assembly of Protein Kinase C α In Vitro.

Protein kinase C α (PKCα) is a nodal regulator in several intracellular signaling networks. PKCα is composed of modular domains that interact with each other to dynamically regulate spatial-temporal function. We find that PKCα specifically, rapidly and reversibly self-assembles in the presence of calcium in vitro. This phenomenon is dependent on, and can be modulated by an intramolecular interaction between the C1a and C2 protein domains of PKCα. Next, we monitor self-assembly of PKC-mCitrine fusion proteins using time-resolved and steady-state homoFRET. HomoFRET between full-length PKCα molecules is observed when in solution with both calcium and liposomes containing either diacylglycerol (DAG) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Surprisingly, the C2 domain is sufficient to cluster on liposomes containing PI(4,5)P2, indicating the C1a domain is not required for self-assembly in this context. We conclude that three distinct clustered states of PKCα can be formed depending on what combination of cofactors are bound, but Ca2+ is minimally required and sufficient for clustering.

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency.

The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase Cα and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.

Selective Modulation of Protein Kinase C α over Protein Kinase C ε by Curcumin and Its Derivatives in CHO-K1 Cells.

Members of the protein kinase C (PKC) family of serine/threonine kinases regulate various cellular functions, including cell growth, differentiation, metabolism, and apoptosis. Modulation of isoform-selective activity of PKC by curcumin (1), the active constituent of Curcuma L., is poorly understood, and the literature data are inconsistent and obscure. The effect of curcumin (1) and its analogues, 4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl oleate (2), (9Z,12Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12-dienoate (3), (9Z,12Z,15Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12,15-trienoate (4), and (1E,6E)-1-[4-(hexadecyloxy)-3-methoxyphenyl]-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (5), and didemethylcurcumin (6) on the membrane translocation of PKCα, a conventional PKC, and PKCε, a novel PKC, has been studied in CHO-K1 cells, in which these PKC isoforms are endogenously expressed. Translocation of PKC from the cytosol to the membrane was measured using immunoblotting and confocal microscopy. 1 and 6 inhibited the TPA-induced membrane translocation of PKCα but not of PKCε. Modification of the hydroxyl group of curcumin with a long aliphatic chain containing unsaturated double bonds in 2-4 completely abolished this inhibition property. Instead, 2-4 showed significant translocation of PKCα but not of PKCε to the membrane. No membrane translocation was observed with 1, 6, or the analogue 5 having a saturated long chain for either PKCα or PKCε. 1 and 6 inhibited TPA-induced activation of ERK1/2, and 2-4 activated it. ERK1/2 is the downstream readout of PKC. These results show that the hydroxyl group of curcumin is important for PKC activity and the curcumin template can be useful in developing isoform specific PKC modulators for regulating a particular disease state.

5-(Bis(3-(2-hydroxyethyl)-1H-indol-2-yl)methyl)-2-hydroxybenzoic acid (BHIMHA): showing a strategy of designing drug to block lung metastasis of tumors.

Early metastasis is still the most recalcitrant factor in the treatment of lung cancer patients. By analyzing the structures and comparing the docking scores of the known pharmacophores, the authors of this paper designed 5-(bis(3-(2-hydroxyethyl)-1H-indol-2-yl)methyl)-2-hydroxybenzoic acid (BHIMHA) as a promising lead compound to develop metastasis inhibitors. In vitro 5, 10, and 20 µM of BHIMHA concentration dependently inhibited the migration and invasion of A549 cells. In vivo 0.4, 2.0, and 8.9 µmol/kg of BHIMHA dose dependently inhibited the metastasis of LLC (Lewis Lung Carcinoma) toward lung. In vivo, 2 µmol/kg of BHIMHA showed additional actions of slowing the growth of the primary tumor of C57BL/6 mice and S180 mice as well as inhibiting xylene-induced ear edema of the mice. Therefore, BHIMHA simultaneously blocked tumor metastasis toward lung, slowed the primary tumor growth, and limited the inflammation. These pharmacological actions were correlated with the inhibition of PKCα and NF-κB expression.