Effects of Ortho Substituent Groups of Protocatechualdehyde

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Effects of Ortho-Substituent Groups of Protocatechualdehyde Derivatives on Binding to the C1 Domain of Novel Protein Kinase C Narsimha Mamidi, Rituparna Borah, Narayan Sinha, Chandramohan Jana, and Debasis Manna J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp304787j • Publication Date (Web): 03 Aug 2012 Downloaded from http://pubs.acs.org on August 7, 2012

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The Journal of Physical Chemistry

Effects of Ortho-Substituent Groups of Protocatechualdehyde Derivatives on Binding to the C1 Domain of Novel Protein Kinase C Narsimha Mamidi, Rituparna Borah, Narayan Sinha, Chandramohan Jana and Debasis Manna Department of Chemistry, Indian Institute of Technology Guwahati, Assam 781039, India *Corresponding author: Tel: 03 61258 2325; Fax: 03 61258 2350; E-mail: [email protected]

Abstract Diacylglycerol (DAG) regulates a broad range of cellular functions including tumor promotion, apoptosis, differentiation and growth. Thus, DAG-responsive C1 domain of protein kinase C (PKC) isoenzymes is considered as an attractive drug target for the treatment of cancer and other diseases. To develop effective PKC regulators, we conveniently synthesized hydroxymethylphenyl ester analogs targeted to the DAG binding site within the C1 domain. Biophysical studies and molecular docking analysis showed that hydroxymethyl group, hydrophobic side chains and acyl group at the ortho position are essential for their interactions with the C1 domain backbone. Modifications of these groups showed diminished binding to the C1 domain. The active hydroxymethyl-phenyl ester analogs showed more than 5 fold stronger binding affinity for C1 domain than DAG. Therefore, our findings reveal that hydroxymethyl-phenyl ester analogs represent an attractive group of C1 domain ligands that can be further structurally modified to improve its binding and activity.

Keywords: Protein kinase C regulators, diacylglycerols, molecular docking analysis, Anisotropy, fluorescence resonance energy transfer, competitive binding

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Introduction The protein kinase C (PKC) family of enzymes is involved in intracellular signal transduction cascades and in various cellular events such as proliferation, differentiation, metabolism and apoptosis. PKC isoenzymes belong to the larger subclass of protein kinases termed the adenineguanine-cytosine (AGC) kinases and serine/threonine kinases. PKC isoenzymes play an important role in pathology of several diseases including cancer, neurological, immunological, cardiovascular and Alzheimer’s diseases. Therefore, PKC isoenzymes have been a subject of intensive research and drug development.1,2 The PKC isoenzymes are activated by sn-1,2-diacyl-glycerols (DAGs) in presence of anionic phospholipids and transmit their signal by phosphorylating specific proteins. The PKC enzyme family consists of at least eleven known isoforms, categorized into conventional (calcium, DAG and phospholipid-dependent), novel (calcium-independent, but DAG and phospholipids dependent) and atypical (calcium and DAG-independent) subgroups. Mammalian cells generally contain low concentration of DAGs under equilibrium conditions.1,3 DAGs are mostly generated by the phosphatidylinositol-specific phospholipase C (PI-PLC)-catalyzed hydrolysis of phosphatidylinositol-4,5 bisphosphate (PtdIns(4,5)P2) at the plasma membrane. The other product of PtdIns(4,5)P2 hydrolysis, inositol-1,4,5-triphosphate ((1,4,5)IP3) releases Ca2+ from endoplasmic reticulum (ER), which also activate various proteins, including Ca2+dependent PKC isoenzymes.4-6 The DAG molecules are also produced from phosphatidylcholine (PC) by concerted action of phospholipase D (PLD) and phosphatidic acid phosphohydrolase, presumably at the internal membranes, such as ER and Golgi membranes.3 The DAG molecules selectively interact with proteins containing a C1 domain. This interaction induces their translocation to the discrete subcellular compartments. For some of the C1 domain containing


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protein, such translocation leads to activation. Other families of signaling proteins like Ras guanyl nucleotide-releasing protein (RasGRPs), chimerins, protein kinase D (PKD), Unc-13 scaffolding proteins, myotonic dystrophy kinase-related Cdc42-binding kinases (MRCK), and the DAG kinases β and γ also shares C1 domain with PKC isoenzymes.7-9 The catalytic domain of PKC isoenzymes is substantially homologous among the protein kinases. The sizes of the C1 domains are small, overall structures are highly conserved and there is only one ligand binding site. Also, the number of C1 domain containing proteins is considerably smaller than the number of protein kinases. Several studies already reported that the regulatory domain of PKCs may have independent biological functions. Therefore, C1 domains have become an attractive target in designing selective PKC ligands.10-13 The majority of high-affinity C1 domain ligands are structurally rigid and complex natural products such as phorbol esters, bryostatins.14,15 Recently developed novel PKC isoform specific indolactam, and benzolactam, derivatives are also highly complex in their structure. Thus, structural modification with the aim of altering the ligand specificity is difficult and largescale production may be unfeasible. Therefore, there is clear and unmet need to design simple surrogates, whose structure could be easily modified to achieve higher specificity and selectivity among the C1 domains of the PKC isoenzymes. Conformationally-constrained DAG lactones, the major groups of selective activators of novel PKC isoenzymes, are considerably simpler than natural products.16-18 Recently reported simple isophthalic acid,2,19 curcumin20 and diacyltetrol derivatives21 have shown stronger affinity for the C1 domain of PKC isoenzymes. In this context, the present study describes the design, synthesis and in vitro binding properties of hydroxymethyl-phenyl ester analogues to the C1b subdomains of PKCδ and PKCθ. The active hydroxymethyl-phenyl ester analogues can compete with DAG binding to C1



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domains of PKC. The hydroxylmethyl group and suitable hydrophobic ester group of the compounds plays an important role in recognizing C1 domains of PKC isoenzymes.

Results and Discussion Design and Synthesis The crystal structure of the PKCδ C1b in complex with phorbol-13-O-acetate shows that hydroxyl and carbonyl groups of the phorbol ester are mainly responsible for its interaction with the C1 domain.22 The hydroxyl groups attached to C-20, C-4 and the carbonyl group on C-3 of the phorbol ester forms hydrogen bonds either with the backbone amide proton or carbonyls. According to structure-activity studies, the C-13 carbonyl group and C-9 hydroxyl group are also important for binding of phorbol-13-O-acetate to the C1 domain. The structure-activity relationship (SAR) studies and biological activities suggest that recently reported hydrophobic derivatives of isophthalic acid, also act as potential C1 domain ligands. The molecular docking analysis of the isophthalic acid derivatives shows similar interaction with the PKCδ C1b subdomain, as the phorbol esters and DAGs. The hydroxyl group and one of the carbonyl groups of dipentyl 5-(hydroxylmethyl)isophthalate form hydrogen bonds with the PKCδ C1 domain.2,19 Whereas, the second carbonyl group of this isophthalate derivative show no direct interactions with the C1 domain. It is proposed that the complex may be stabilized by either through a bridging water molecule or by interaction with anionic phospholipids.2 The hydrophobic side chains of the C1 domain lignds, including phorbol esters, DAGs and isophthalic acid derivatives are reported to interact either with the hydrophobic amino acids (Met-239, Phe-243, Leu-250, Trp 252, and Leu-254) surrounding the ligand binding cleft or with the hydrophobic moiety in the lipid bilayer.17,22


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We selected hydroxymethyl-phenyl ester analogues for the development of C1 domain based PKC modulator. These compounds can be synthesized from the corresponding protocatechualdehyde, vanillin, isovanillin and are easily derivatizable. These aldehydes are secondary plant metabolites and have many pharmacological effects, including antioxidant, antitumor activity and others.23 The two hydroxyl groups of protocatechualdehyde provide the access to incorporate different functionalities. It also allows incorporating two of the phorbol ester pharmacophores (hydroxyl and the carbonyl functionalities) within the same interatomic distance as in phorbol ester. The long alkyl chain attached to the hydroxyl group at the first position is useful for anchoring the ligands in the lipid bilayer/membranes and for interaction with anionic phospholipids. The second hydroxyl group is available to introduce different functionalities for optimization of the protein binding. The hydrophobic interactions are difficult to model. Thus, we synthesized a series of hydroxymethyl-phenyl ester analogues with different side chains to study the impact that different functionality and side chains had on the binding affinity (Table 1). We initially synthesized 4-hydroxymethyl phenyl diester analog (5) in two steps using commercially available protocatechualdehyde as the starting material (Scheme 1). The compound 5, with long (palmitic acid) and short (caprylic acid) chain fatty acids were synthesized to study the impact of alkyl chain length on the binding affinity. To investigate the importance of two ester groups with alkyl chains and interatomic distance between the phorbol ester pharmacophores in C1b subdomain binding, we prepared 3-hydroxymethyl phenyl ester (3) and 4-hydroxymethyl phenyl ester (4), with mono-ester group at meta- and para-position, respectively.



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Table 1. Structure of the synthesized compounds used for the present study. R3 R2

R1

1a 1b 2a 2b 3a 3b 4a 4b 5a 5b 6 7a

R1 OH OH OH OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH

Compound R2 H H OCO(CH2)14CH3 OCO(CH2)6CH3 H H OCO(CH2)14CH3 OCO(CH2)6CH3 OCO(CH2)14CH3 OCO(CH2)6CH3 OCO(CH2)14(CH)2CH3 OCOCH3

R3 OCO(CH2)14CH3 OCO(CH2)6CH3 H H OCO(CH2)14CH3 OCO(CH2)6CH3 H H OCO(CH2)14CH3 OCO(CH2)6CH3 OCO(CH2)14(CH)2CH3 OCO(CH2)14CH3

1-13

7b 8a 8b 9a 9b 10a 10b 11a 11b 12a 12b 13

R1 CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH CH2OH

Compound R2 OCOCH3 OCOC6H5 OCOC6H5 OCH3 OCH3 OCO(CH2)14CH3 OCO(CH2)6CH3 OC(CH3)3 OC(CH3)3 OCH2C6H5 OCH2C6H5 OCO(CH2)10(CH)8CH3

R3 OCO(CH2)6CH3 OCO(CH2)14CH3 OCO(CH2)6CH3 OCO(CH2)14CH3 OCO(CH2)6CH3 OCH3 OCH3 OCO(CH2)14CH3 OCO(CH2)6CH3 OCO(CH2)14CH3 OCO(CH2)6CH3 OCO(CH2)10(CH)8CH3

To investigate the importance of the hydroxymethyl group of the compounds 3-5, we prepared hydroxyphenyl ester analogs 1 and 2, with required phorbol ester pharmacophores. It is also reported that DAGs with unsaturated ester groups generated by PI-PLC are more potent in PKC activation than saturated forms produced by other pathways.3,24-26 We therefore, prepared 4hydroxymethyl phenyl diester analog 6 and 13 with unsaturated fatty acid (oleic acid and arachidonic acid, respectively). To understand the role of long alkyl chain in the ortho-position, we also prepared compound 7 and 8 with acetic acid and benzoic acid, respectively. The ether group was also introduced to understand the importance of ester group at ortho-position in C1 domain binding. The position of ester group and ether group of 9 and 10 were selected for further SAR studies. To increase the hydrophobicity of the ether-linked side chain, we prepared compound 11 and 12 with tert-butyl and benzyl ether groups respectively.


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OH OH

Palmitic acid/ Octanoic acid, DCC, DMAP

77-90%

NaBH4, THF

OR

00C-rt, 20- 30min 80-96%

CH2Cl2, rt, 12h CHO

OR

OR

CHO

OR

CH2OH 5

5a (R = CO(CH2)14CH3) 5b (R = CO(CH2)6CH3)

Scheme 1. Synthesis route to hydroxymethyl-phenyl ester (5).

Our molecular docking analysis shows that the hydroxymethyl-phenyl ester analogs are anchored to binding site of the C1 domain of PKC in a similar fashion, as phorbol esters, DAGlactones and dialkyl 5-(hydroxylmethyl)isophthalate.2,22,27 The ligand’s hydroxyl group is also hydrogen bonded to the backbone amide proton of Thr-242 and the carbonyls of Thr-242 and Leu-251 (Figure 1). The model structure also showed that one carbonyl group of the ligand 5b, 7b and 8b is also hydrogen bonded with the backbone amide proton of Gly-253. The other carbonyl group may be involved in interactions with charged lipid head groups, including phosphatidylserine for activation.28 Whereas, the hydrogen bond with the backbone amide proton of Gly-253 is missing for ligand 9b-12b, due the presence of ether group (Figure S1). Hydroxyphenyl ester analogs 1 and 2 were directly synthesized from the corresponding dihydroxybenzene, using standard DCC-mediated coupling reaction with octanoic acid and palmitic acid.29 The hydroxymethyl-phenyl ester analogues (3-6, 13) were conveniently synthesized in two steps using the commercially available corresponding hydroxybenzaldehydes as the starting material. The phenolic hydroxyl groups of the hydroxybenzaldehydes were esterified with octanoic acid, palmitic acid, oleic acid and arachidonic acid using DCC-mediated coupling reaction to produce formyl-phenyl ester analogs. Subsequent reduction of the formyl group with NaBH4 provided the target hydroxymethyl-phenyl ester analogues 3-6 and 13. For the 7 ACS Paragon Plus Environment


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preparation of compound 7, 8, 11 and 12, we first synthesized 4-formyl-2-hydroxyphenyl octanoate/hexadecanoate from protocatechualdehyde using DCC-mediated coupling reaction

Figure 1: Structures of ligand bound PKCδ C1b subdomain. (A) Crystal structure of phorbol 13O-acetate bound PKCδ C1b; (B) modeled structure of 5b docked into PKCδ C1b; (C) modeled structure of 8b docked into PKCδ C1b. The modeled structures were generated using the Molegro Virtual Docker, version 4.3.0. The oxygen atoms and nitrogen atoms are shown in red and blue, respectively. The dotted line indicates possible hydrogen bonds.

with octanoic acid and palmitic acid. The selective formations of these monoesters were characterized by NMR and Mass spectral analysis. Further DCC-mediated coupling reaction with acetic acid/benzoic acid and subsequent reduction of formyl group with NaBH4 resulted compound 7 and 8. Treatment of 4-formyl-2-hydroxyphenyl octanoate/hexadecanoate with tertbutyl iodide/benzyl bromide using Ag2O and reduction of formyl group with NaBH4 yielded compound 11 and 12. The compound 9 and 10 were prepared from vanillin and isovanillin, respectively via DCC-mediated esterification followed by reduction of formyl group with 8 ACS Paragon Plus Environment

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NaBH4.

Protein binding In the classical and novel PKC isoenzymes, the DAG-sensitive C1 domain is duplicated into a tandem C1 domain consisting of C1a and C1b subdomains. The C1b subdomains of PKCδ and PKCθ are reported to have sufficiently stronger DAG binding affinity and easy to obtain them in soluble form in sufficient amount from bacterial cells. In the present study, these C1b subdomains were used to measure in-vitro binding potencies of the synthesized compounds in monomeric form by using intrinsic fluorescence quenching method. We also used Förster Resonance Energy Transfer (FRET) based liposome binding assay and competitive displacement assay to measure the ligand binding affinity and specificity under liposomal environment.30,31

Interaction with soluble ligands The intrinsic fluorescence of the PKC C1b subdomains is due to the presence of a single Tryptophan (Trp-252 in delta, and Trp-253 in theta) and Tyrosine residues (Tyr-236 and Tyr-238 in delta, Tyr-249 and Tyr-251 in theta) and the change in its conformation or microenvironment caused by ligands can be detected by fluorescence spectroscopy. The single Trp residue is also present close to the DAG binding pocket of the C1b subdomains of PKCδ and PKCθ.20,21 Figure S2 shows the representative plot of fluorescence quenching data for PKCδ C1b in the presence of ligands. The measured binding affinities revealed that compound 5, 6, 8 and 13 (Figure 2) with different chain length interact with the C1b subdomains with highest affinity (0.54–1.15 μM) and



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Figure 2: Binding of ligands with PKCδ C1b. Representative plot of fluorescence intensity of PKCδ C1b (2 μM) in buffer (20 mM Tris, 160 mM NaCl, 50 μM ZnSO4, pH 7.4) in the presence of varying concentration of 5b (●), 7b (◊), 8b (■), 11b (♦), 12b (◘) and 13 (∆), where F and F0 are fluorescence intensity in the presence and absence of the ligands, respectively. The solid lines are nonlinear least squares best fit curves.

other compounds had comparable binding affinity for both the proteins (Table 2). Monomeric ligands 5b and 8b show more than 5 fold stronger binding affinity than DAG8 for PKCδ protein. The binding results also show that hydroxymethyl-phenyl esters have higher binding affinity for PKCθ C1b as compared to the PKCδ C1b, possibly due to the additional hydrogen bonds. The molecular models of ligand bound protein showed that, there were three possible hydrogen bonds among DAG and PKCδ C1b, whereas compound 5b and 8b showed four hydrogen bonds with PKCδ C1b. The experimental binding results and docking score values obtained from the models do not always corroborate (Table S1). This difference indicates that both proteins and compounds can undergo conformational changes under experimental conditions. 10 ACS Paragon Plus Environment

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To understand the importance of hydrophobicity of the compounds in C1 domain binding, similar analysis was performed with the long chain hydroxymethyl-phenyl esters. Both proteins showed a similar pattern of dependence on the hydrophobicity of the compounds (Table 2). For compound 8a (XLOGP3 = 9.46) and 8b (XLOGP3 = 5.13), although there is a distinct difference in hydrophobicity, but the difference in binding affinity among them are very small for both the proteins. This could be due to the binding orientations with the C1b subdomains through the hydroxymethyl group. The molecular docking analysis also indicates that the overall high binding affinities of the compounds 5 and 8 are probably associated with the presence of hydroxymethyl and the carbonyl functionalities within the same interatomic distance as in phorbol ester. The hydrophobic amino acids, surrounding the binding site of the C1 domain also interact with the hydrophobic side chains of the ligands. Thus, the binding affinity values of hydroxymethyl-phenyl esters highlight the importance of ligand hydrophobicity and binding orientation, in similar manner to those reported for C1 domain ligands.

Table 2. KD (ML) values for the binding of ligands with the PKCδ C1b and PKCθ C1b proteinsa at room temperature. Compound

DAG16 1a 2a 3a 4a 5a 6 7a 8a 9a 10a 11a 12a a)

KD (ML) / (μM) PKCδ C1b 7.02 ± 0.33 6.17 ± 0.26 5.83 ± 0.28 5.13 ± 0.31 4.78 ± 0.31 1.10 ± 0.09 1.03 ± 0.07 1.27 ± 0.08 0.91 ± 0.15 1.98 ± 0.19 3.45 ± 0.18 1.84 ± 0.11 1.67 ± 0.15

PKCθ C1b 7.47 ± 0.48 1.07 ± 0.05 1.26 ± 0.11 0.81 ± 0.08 1.59 ± 0.09 1.62 ± 0.03 1.71 ± 0.05

XLOGP3

Compound

14.04 8.64 8.56 8.11 8.11 15.19 14.95 7.87 9.46 8.09 8.09 9.07 9.35

DAG8 1b 2b 3b 5b 7b 8b 9b 10b 11b 12b 13

KD (ML) / (μM) PKCδ C1b 10.17 ± 0.68 7.25 ± 0.49 6.94 ± 0.37 5.26 ± 0.28 1.23 ± 0.17 1.33 ± 0.13 1.02 ± 0.16 2.32 ± 0.19 3.87 ± 0.16 1.87 ± 0.13 1.96 ± 0.18 0.83 ± 0.09

XLOGP3 PKCθ C1b 6.29 ± 0.44 1.17 ± 0.11 1.27 ± 0.13 0.99 ± 0.13 1.98 ± 0.17 1.74 ± 0.22 1.84 ± 0.11 0.54 ± 0.12

5.11 4.31 4.22 3.78 6.53 3.53 5.13 3.75 3.75 4.74 5.02 13.54

Protein, 1 μM in buffer (20 mM Tris, 160 mM NaCl, 50 μM ZnSO4, pH 7.4)



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To gain more information about ligand-protein interactions, we performed steady-state fluorescence anisotropy measurements of the proteins in absence or presence of the ligands and DAGs. The increase in anisotropy values of the proteins in presence of ligands support their ligand binding. The degree anisotropy of pure PKCδ C1b protein increases from 0.0439 in buffer to 0.0874 and 0.0850 on interactions with 10-fold excess of ligands 5b and 8b, respectively (Table 3). Similar increase in anisotropy values were observed for proteins in presence of DAGs and other compounds.

Although the changes in anisotropy values were different for the

compounds, still this experiment suggested that presence of compounds increase the rigidity of the surrounding environment of the protein in a similar manner to that of DAGs.

Interaction with ligand associated liposomes Peripheral proteins like PKCs are reported to interact with the membrane surface through their lipid-binding C1 and C2 domains. The C1 domains have both membranes binding surface, and lipid-binding groove. The C1 domain responds to increased DAG levels at the plasma membrane. To measure the binding properties of the C1b subdomains of PKC isoforms with the long chain hydroxymethyl-phenyl ester analogs, we performed protein-to-membrane FRET based liposome binding assay and competitive binding assay. In this assay, one Trp residue in the C1b subdomains of PKC serve as FRET donor and a low density of membrane-embedded, dansyl-PE (dPE) lipids serve as acceptors.30,31 FRET based liposome binding assay was employed to compare the qualitative ligand selectivity of the C1b subdomains under liposomal environment. The protein-to-membrane FRET signal was monitored to measure the protein docking to the ligand-associated liposome


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surface. In a separate experiment, liposomes without the ligands were titrated into a solution of protein, to control the increasing background emission arising from direct dansyl-PE excitation and PS binding of C1b subdomains. Following subtraction of background signal and correction for dilution, the binding isotherm was generated. The qualitative ligand selectivity pattern shows that C1b subdomains have better binding affinity for 5a, 6 and 8a-associated liposomes than DAG16-associated liposomes, under similar experimental condition (Table 4). Ligands 5a, 6 and 8a liposomes show more than 5 fold stronger binding affinity than DAG16.

Such titration

experiments are useful for measuring relative binding affinities under similar liposomal environment, but not for accurate determination of affinities. The dansyl-PE concentration needed for the FRET assay significantly exceeds the binding constant of the interaction.

Table 3. Anisotropya values of the ligands in the presence and absence of the PKCδ and PKCθ C1b proteins at room temperature. Compound Bufferb DAG16c 1ac 2ac 3ac 4ac 5ac 6c 7ac 8ac 9ac 10ac 11ac 12ac a)

PKCθ C1b 0.0667 (0.0073) 0.0727 (0.0037) 0.0984 (0.0156) 0.0973 (0.0025) 0.0903 (0.0028) 0.0946 (0.0006) 0.1146 (0.0025) 0.1317 (0.0016)

Compound DAG8c 1bc 2bc 3bc 4bc 5bc 7bc 8bc 9bc 10bc 11bc 12bc 13c

PKCδ C1b 0.0978 (0.0063) 0.0596 (0.0077) 0.0856 (0.0013) 0.0562 (0.0032) 0.0660 (0.0026) 0.0874 (0.0161) 0.0565 (0.0042) 0.0850 (0.0119) 0.0834 (0.0047) 0.0673 (0.0038) 0.0766 (0.0029) 0.0722 (0.0031) 0.1001 (0.0028)

PKCθ C1b 0.1069 (0.0051) 0.1111 (0.0046) 0.0884 (0.0005) 0.0896 (0.0022) 0.0933 (0.0008) 0.0945 (0.0016) 0.0950 (0.0044) 0.0998 (0.0012)

Values in the parenthesis indicate standard deviations.

b)

c)

PKCδ C1b 0.0439 (0.0045) 0.1028 (0.0061) 0.1424 (0.0026) 0.0994 (0.0063) 0.0892 (0.0045) 0.0768 (0.0032) 0.0685 (0.0065) 0.0915 (0.0008) 0.0862 (0.0051) 0.0838 (0.0077) 0.0889 (0.0075) 0.1156 (0.0132) 0.1217 (0.0024) 0.1129 (0.0055)

Protein, 1 μM in buffer (20 mM Tris, 160 mM NaCl, 50 μM ZnSO4, pH 7.4).

DAG, 1-13, 10 μM; protein, 1 μM in buffer (20 mM Tris, 160 mM NaCl, 50 μM ZnSO4, pH 7.4).

We used FRET based competitive binding assay to compare quantitatively the binding



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affinity and specificity of C1b subdomains for the liposome-associated targeted ligand. In this method competitive inhibitor DAG8 was titrated into the solution containing C1b subdomain bound liposomes. The decrease in the protein-to-membrane FRET signal (Figure S3) was monitored to measure the displacement of protein from liposomes by DAG8 and to calculate apparent inhibitory constant (KI(DAG8)app). Figure 3 represents DAG8 induced competitive displacement of C1b subdomain from 5% targeted ligand associated liposomes. The apparent inhibition constant (KI(DAG8)app) for DAG8 depends on ligand concentration in the liposomes and the background lipid composition as well as the affinities of the C1b subdomains for ligands and DAG8. This assay confirmed that the synthesized ligands interact with the C1 domains through the DAG/phorbol ester binding site. The results also showed that higher concentration of DAG8 was required for the displacement of both the proteins from the ligands 5a, 6, 8a and 13 associated liposomes (Table 4).

Table 4: Equilibrium parameters for PKCδ C1b and PKCθ C1b proteina binding to the ligand associated liposomesb at room temperature. Compound 5a 6 7a 8a 11a 12a 13 DAG16 a)

KD (LL) / (nM) PKCδ C1b PKCθ C1b 3.36 ± 0.58 2.80 ± 0.19 3.24 ± 0.64 2.76 ± 0.11 6.40 ± 0.45 3.53 ± 0.34 2.87 ± 0.27 2.53 ± 0.21 9.06 ± 0.61 6.98 ± 0.60 6.76 ± 0.30 4.64 ± 0.89 2.98 ± 0.28 2.19 ± 0.18 17.13 ± 0.98 14.70 ± 1.34

KI (DAG8)app / (μM) PKCδ C1b PKCθ C1b 23.43 ± 1.03 21.54 ± 0.69 23.85 ± 1.85 23.22 ± 1.22 19.79 ± 1.05 13.85 ± 0.54 28.02 ± 1.82 24.11 ± 1.77 13.13 ± 0.90 12.02 ± 1.54 14.14 ± 01.75 12.91 ± 1.79 30.14 ± 2.22 25.79 ± 1.93

-

KD(L16) / (nM) PKCδ C1b PKCθ C1b 129.56 ± 5.51 137.48 ± 3.96 117.78 ± 3.11 183.47 ± 5.23 267.76 ± 6.78 89.29 ± 3.11 92.49 ± 2.19 434.85 ± 9.14 415.89 ± 6.98 356.76 ± 4.59 406.87 ± 10.15 75.33 ± 1.87 59.02 ± 2.59

-

Protein, 1 μM in buffer (20 mM Tris, 150 mM NaCl, 50 μM ZnSO4, pH 7.4).

b)

Active liposome composition, PC/PE/PS/dPE/Ligand16 (55/15/20/5/5).

Finally, the equilibrium dissociation constant (KD(L16)) for the C1b subdomain binding to the liposome-associated targeted ligand was calculated using the standard equation for 14 ACS Paragon Plus Environment

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competitive inhibition.31 Comparison of the equilibrium dissociation constant for competitive inhibitor DAG8 reveals that C1b subdomains have higher binding affinities for the compound 5, 6, 8 and 13 associated liposomes (Table 4). The molecular docking and binding analysis shows that the synthesized ligands interact differentially with C1b subdomain of PKCδ and PKCθ, both in monomeric form and under liposomal environment. Modifications of the ester side chains have a modest effect on the binding. The compounds 5–8 and 13 have both the phorbol ester pharmacophores. But, the compound 7 with shorter ester group at the ortho-position shows weaker binding affinity. These results highlight the similar importance of hydrophobicity in the ligands as those for other C1 domain ligands. Our studies with compound 9–12 shows that, lack of ester group at the orthoposition contribute to diminished binding affinity. The only difference in functionality at the ortho-position between compound 8 and 12, causing more than 2 fold binding affinity difference



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Figure 3: Competitive displacement assay for the PKCδ C1b subdomains (1 µM) bound to liposome containing ligands 5a (●), 7a (◊), 8a (□), 11a (♦), 12a (◘) and 13 (∆). The bound complex was titrated with the competitive inhibitor DAG8.

for both the proteins under the similar experimental conditions. Our studies with compound 6 and 13 having unsaturated acyl groups, showed higher binding affinity than compound 5 with saturated acyl groups.

The results are in accordance with the reported binding affinity of

unsaturated DAG to C1 domains.25,32 This could be due to either genuine selectivity of PKC C1 domains for ligands with unsaturated acyl groups or the effect of these ligands on the membrane structure.

Extent of membrane localization To evaluate the relationship between the protein binding properties of the ligands and the extent of their localization at the bilayer/water interface, we performed fluorescence quenching experiments using PC/Cholesterol/Ligand16/NBD-PE liposomes. The NBD dye is embedded close to the bilayer interface, providing a useful marker for surface interactions of membraneactive C1 domain ligands. The NBD fluorescence quenching by sodium dithionite provided a measure of membrane interactions of the ligands.18,33 Figure 4 demonstrates that ligand associated fluorescent liposomes yielded significant changes in the rate of dithionite-induced fluorescence quenching of the bilayer-embedded dye. All the examined ligands yielded lower quenching rates compared to the control liposomes (without any ligands). The results suggest that the NBD dye became more ‘shielded’ from the soluble dithionite quencher, due to the presence of hydroxymethyl-phenyl ester analogs in the liposomes. The results also infer that


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these ligands are more localized at the liposome surface compared to DAG. Thus, the hydroxymethyl-phenyl ester analogs (5a, 7a, 8a, 11a and 12a) are more accessible for the protein binding than DAG, under liposome environment. This is in complete correlation with their protein binding properties under the liposome environment.

Figure 4: Fluorescence quenching of NBD-PE embedded in PC/Cholesterol/Ligand16/NBD-PE (44.5:44.5:10:1) liposomes. Sodium dithionite = 0.6 µM. Control: no ligand.

Overall, these results demonstrate that hydroxymethyl group and one ester group with hydrophobic side chain at the ortho position are needed for binding activity of the compounds to the C1 domain, but if the compound has one ether group at ortho position another ether/ester linked hydrophobic side chain is also required. The results also show that the long chain hydroxymethyl-phenyl esters can differentially influence their in vitro membrane interaction properties of PKCθ and PKCδ. The affinity differences between the proteins are solely because



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of the differences in the residues and surface area of the activator binding pockets. The activating effect of hydroxymethyl-phenyl esters can be lower than the phorbol esters or other natural ligands under similar experimental conditions.

Conclusion We described here hydroxymethyl-phenyl ester analogs, which interact with the C1 domain of PKC isoforms through the DAG binding site. Synthesis of these compounds from the corresponding aldehydes provided the ability to generate pure samples and freedom to explore the role of hydroxymethyl group, hydrophobic side chains and ester group at the ortho position. The active compounds can also compete with DAG for binding to C1 domains of PKC under liposomal environment. This makes these compounds, in particular the derivatives of protocatechualdehyde as potential regulator of PKC isoforms and can be further developed as research tools or lead compound in drug development.

Experimental Section Materials and general procedures: All reagents were purchased from Sigma, USA and Merck, India and used directly without further purification. Dry solvents were obtained according to the reported procedures. Column chromatography was performed using 60-120 mesh silica gel. Reactions were monitored by TLC on silica gel 60 F254 (0.25 mm). 1H NMR and

13

C NMR

spectra were recorded at 400 MHz and at 100 MHz respectively, by using a Varian AS400 spectrometer. Coupling constants (J value) were reported in Hertz (Hz). The chemical shifts were shown in parts per million (ppm) downfield from tetramethylsilane, using residual chloroform (δ = 7.24 in 1H NMR, δ = 77.23 in 13C NMR), as an internal standard. Multiplicities were reported


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as follows: s (singlet), d (doublet), t (triplet), m (multiplet) and br (broadened). Melting points were determined using Büchi B-545 melting point apparatus and are uncorrected. Mass spectra were recorded using WATERS MS system, Q-tof premier and data analyzed by using the built-in software. 1,2-dipalmitoyl-sn-glycerol (DiC18), 1,2-dioctanoyl-sn-glycerol (DiC8), 1,2-dioleoylsn-glycero-3-phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl)

(NBD-PE)

were purchased from Avanti Polar Lipids. The N-[5-(dimethylamino)naphthalene-1-sulfonyl]1,2-dihexadecanoyl-sn-glycero-3 phosphoethanolamine (Dansyl-PE) was purchased from Invitrogen, USA. Ultrapure water (Milli-Q system, Millipore, USA) was used for the preparation of buffer.

Synthesis of formylphenyl mono ester analogues: Hexadecanic acid/Octonic acid (1.1 equiv), dicyclohexylcarbodiimide (1.1 equiv) and N, N-dimethylaminopyridine (0.1 equiv) were added to a stirring solution of mono hydroxybenzaldehyde (1.0 equiv) in anhydrous dichloromethane (8 mL) under N2 atmosphere. Stirring was continued for 12 h at room temperature. After completion of reaction (monitored by TLC), the reaction mixture was filtered and washed (3×) with dichloromethane. The filtrate was concentrated under reduced pressure and the column chromatography was performed with silica gel and a gradient solvent system of 0-8% ethyl acetate to hexane to provide corresponding esters. The average yields were 77% to 90%.

Synthesis of formyl-phenyl diester analogues: Palmitic acid/Octonic acid (2.2 equiv), dicyclohexylcarbodiimide (2.2 equiv) and N, N-dimethylaminopyridine (0.1 equiv) were added to a stirring solution of dihydroxybenzaldehyde (1.0 equiv) in anhydrous dichloromethane (8 mL) under N2 atmosphere. Stirring was continued for 12 h at room temperature. After



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completion of reaction, the reaction mixture was filtered and washed (3×) with dichloromethane. The filtrate was concentrated under reduced pressure and the column chromatography was performed with silica gel and a gradient solvent system of 0-8% ethyl acetate to hexane, yielded corresponding esters. The average yields were 77% to 90%.

Reduction of aldehydes: To the stirring solution of formylphenyl ester analogues (1.0 equiv) in THF, NaBH4 (2.0 equiv) was added by portion-wise at 0°C over 5 minutes and stirring was continued for another 15 minutes at room temperature. After completion of the reaction (monitored by TLC), the mixture was cooled to 0°C and carefully quenched by 1 ml of acetic acid, followed by 3 ml of water and extracted with ethylacetate (3×10 ml). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. Column chromatography with silica gel and a gradient solvent system of 10-30% ethyl acetate to hexane yielded corresponding (hydroxymethyl)phenyl esters analogues as final products. The average yields were 80% to 96%.

Synthesis of 2-tert-butoxy and 2-(benzyloxy)-4-formylphenyl hexadecanoate /octanoate: To a solution of 4-formyl-2-hydroxyphenyl hexadecanoate/octanoate (1.0 equiv) in anhydrous dichloromethane (10 mL), silveroxide (1.5 equiv) and benzyl bromide/tert butyl iodide (1.2 equiv) were added under a N2 atmosphere. Stirring was continued for 8 h at room temperature. After completion of the reaction, the reaction mixture was filtered off through a pad of Celite. The solvent was removed under reduced pressure. Column chromatography with silica gel and a gradient solvent system of 6–10% ethyl acetate to hexane yielded target compounds. The average yields were 80% to 92%.


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Compound characterization: Detailed descriptions of compound characterizations are provided in the Supporting Information.

Protein purification: The PKCδ and PKCθ C1b subdomains were expressed in E. coli as a GST-tagged protein, purified by glutathione sepharose column and the GST tag was removed by the thrombin treatment using similar methods described earlier.20,21,33,34

Fluorescence measurements: Fluorescence measurements were performed on Fluoromax-4 spectrofluorometer at room temperature. To study the binding parameters under membrane free system, the stock solutions of compounds were freshly prepared by first dissolving complexes in spectroscopic-grade dimethylsulfoxide (DMSO) and then diluted with buffer. The amount of DMSO was kept less than 3% (by volume) for each set of experiment and had no effect on any experimental results. For fluorescence titration, protein (1 μM) and varying concentration of ligands were incubated in a buffer solution (20 mM Tris, 150 mM NaCl, 50 μM ZnSO4, pH 7.4) at room temperature. Protein was excited at 284 nm and emission spectra were recorded from 300 to 550 nm. Proper background corrections were made to avoid the contribution of buffer and dilution effect. The resulting plot of intrinsic fluorescence as a function of ligand concentration was subject to nonlinear least-squares best-fit analysis to calculate the apparent dissociation constant for ligands (KD(ML)), using equation 1, which describes binding to a single independent site. (

− )= ∆

[ ] [ ]

(

)

+

(1)



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Where, F and F0 represented the fluorescence intensity at 339 nm in the presence and in the absence of ligand respectively. The ΔFmax represents the calculated maximal fluorescence change, [x] represents the total monomeric ligand concentration. Fluorescence anisotropy measurements were also performed on the same fluorimeter using similar methods described earlier.21 All anisotropy values of the proteins in the absence or presence of compounds are the mean values of three individual determinations. The degree (r) of anisotropy in the tryptophan fluorescence of the proteins was calculated using equation 2, at the peak of the protein fluorescence spectrum, where IVV and IVH are the fluorescence intensities of the emitted light polarized parallel and perpendicular to the excited light, respectively, and G = IVH/IHH is the instrumental grating factor. =

(

)

(

)

(2)

Analysis of protein-to-membrane FRET for PKC C1b subdomain bound to the ligands associated liposomes was carried out by titrating vesicles composed of PC/PE/PS/dPE (60/15/20/5), PC/PE/PS/dPE/Ligand16 (55/15/20/5/5) with the C1b subdomain. The liposomes were prepared according to the described method in a buffer solution (20 mM Tris, 150 mM NaCl, 50 μM ZnSO4, pH 7.4). The liposomes were titrated into a solution containing C1b suddomain and the protein-to-membrane FRET was measured from the dPE emission (λex = 284 nm and λem = 522 nm). In a separate sample, identical vesicles were added to buffer lacking protein to control for the increasing background emission arising from direct dPE excitation. Following subtraction of the background emission and correction for dilution, the FRET titration curve was best-fit with equation 3.

(

− )= ∆

[ ] [ ]

(

)

+

(3) 22

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Where, ΔFmax represents the calculated maximal fluorescence change, [x] represents the total liposome bound ligand concentration and KD(LL) represents the apparent dissociation constant for ligand binding under liposome environment. FRET based competitive binding assay was used to quantitatively compare the affinity and specificity of the selected ligands under liposomal environment. In this assay membrane bound C1b subdomain was displaced from vesicles by the addition of the competitive inhibitor DAG8. The stock solution of DAG8 was titrated into the sample containing C1b subdomain (1 μM) and excess liposome (100 μM total lipid) in a buffer solution (20 mM Tris, 150 mM NaCl, 50 μM ZnSO4, pH 7.4) at room temperature. The competitive displacement of C1b subdomain from the membrane was quantitated using protein-to-membrane FRET signal. A control experiment was performed to measure the dilution effect under similar experimental condition. Plots of protein-to-membrane FRET as a function of DAG8 concentration were subjected to nonlinear least-squares-fit analysis using equation 4 to calculate apparent equilibrium competitive inhibition constants (KI(DAG8)app) for DAG8. Where, [x] represents the total DAG8 concentration and ΔFmax represents the calculated maximal fluorescence change. = ∆

1−[

[ ] ]

(

)

+

(4)

The equilibrium dissociation constant (KD(L16)) for the binding of the C1 domains to the ligandassociated liposomes was calculated from equation 5, using KD(ML) and KI(DAG8)app values. Where, [L16]free is the free ligand concentration (2.65 ± 0.04 μM). In this assay ligands in the liposome interior were ignored, since it cannot be accessed by the protein. Thus, about half of the lipids in the liposomes are accessed by the protein. The ligand concentration was taken excess relative to the protein. The free ligand concentration was calculated by assuming that most of the



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protein would bind to the liposome and equimolar amount of ligand can be subtracted from the accessible ligand.30,31 (

)

=

(

) 1+

[

] (

(5)

)

Extent of membrane localization: The extent of localization of the ligands at the liposome interface was studied by fluorescence quenching method, using PC/Cholesterol/Ligand16/NBDPE liposomes (44.5/44.5/10/1) in 50 mM Tris buffer, pH 8.2, containing 150 mM NaCl, according to the reported procedure.18,33

Molecular modeling: Molecular docking modeling was performed using the crystal structure of PKCδ C1b (Protein Data Bank code: 1PTR)22. The generation of energy minimized threedimensional structure of ligands and ligand-protein docking was performed using the similar methods described earlier.21,33

Acknowledgments The

authors

are

thankful

to

the

BRNS

(2009/20/37/5/BRNS/3331)

and

DBT

(BT/PR13309/GBD/27/244/2009), Govt. of India, for financial support.

Supporting Information Available Characterization data of the synthesized compounds. Liposome binding assay and extent of membrane localization studies of the compounds. Molecular docking results of the compounds. Copies of 1H, 13C NMR and MS spectra of new compounds. This information is available free of charge via the Internet at http://pubs.acs.org.


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Effects of Ortho-Substituent Groups of Protocatechualdehyde Derivatives on Binding to the C1 Domain of Novel Protein Kinase C Narsimha Mamidi, Rituparna Borah, Narayan Sinha, Chandramohan Jana and Debasis Manna Department of Chemistry, Indian Institute of Technology Guwahati, Assam 781039, India *Corresponding author: Tel: 03 61258 2325; Fax: 03 61258 2350; E-mail: [email protected]

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Effects of Ortho Substituent Groups of Protocatechualdehyde

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