Membrane Interaction and Protein Kinase C‑C1 Domain Binding Properties of 4‑Hydroxy-3-(hydroxymethyl) Phenyl Ester Analogues Dipjyoti Talukdar, Subhankar Panda, Rituparna Borah, and Debasis Manna* Department of Chemistry, Indian Institute of Technology, Guwahati, Assam 781039, India * S Supporting Information ABSTRACT: Protein kinase C (PKC)-C1 domain targeted regulator development is considered as a potential therapeutic strategy for the treatment of cancer and immunological and other diseases. Efforts are underway to synthesize small molecules to achieve higher specificity for the C1-domain than the natural activator, diacylglycerols (DAGs). In this regard, we conveniently synthesized 4-hydroxy-3-(hydroxymethyl) phenyl ester analogues and measured in vitro C1-domain binding properties. We also investigated different physico- chemical properties of the synthesized molecules, including aggregation behavior in aqueous solution and interaction with lipid bilayers, and others with an aim for better understanding of their C1-domain binding properties. The results showed that the membrane-active compounds aggregate in aqueous solution at a reasonably lower concentration and strongly interact with the lipid bilayer. The hydrophilic part of the compounds localize at the bilayer/water interface and accessible for C1-domain binding. Biophysical studies revealed that the hydroxyl, hydroxymethyl, and carbonyl groups and acyl chain length are important for their interaction with the C1-domain. The potent compound showed more than 10-fold stronger binding affinity for the C1- domains than DAG under similar experimental conditions. Therefore, our findings reveal that these ester analogues represent an attractive group of C1-domain ligands that can be further structurally modified to improve their binding and activity. ■ INTRODUCTION There are 518 protein kinases present in the human genome that transfers the phosphate group from adenosine-5- triphosphate to other substrate proteins. 1 These protein kinases specifically phosphorylate serine, threonine, and tyrosine amino acids of the target proteins. The phosphorylation process leads to conformational change of the target proteins that trigger signaling cascades, which in turn regulate cell proliferation, differentiation, migration, survival, and apoptosis. 2,3 The protein kinase C (PKC) enzymes belong to the serine/ threonine kinases family. PKC enzymes play an important role in the pathology of several diseases including cancer and neurological, immunological, cardiovascular, and Alzheimer’s diseases. In consequence, PKC enzymes are being actively pursued as the subject of intense research and drug develop- ment. 4-7 The PKC enzymes consist of a highly conserved C-terminal catalytic domain and a less conserved N-terminal regulatory domain, which comprises an autoinhibitory pseudosubstrate sequence and one or two membrane-targeting domains (DAG/ phorbol ester-binding C1-domain and Ca 2+ -binding C2- domain). On the basis of their enzymatic properties and activation mechanism, the mammalian PKC enzymes have been categorized into classical (Ca 2+ -, DAG-, and phospholipid- dependent), novel (Ca 2+ -independent, but DAG- and phos- pholipid-dependent), and atypical (Ca 2+ - and DAG-independ- ent) subgroups. 4,8,9 In eukaryotic cells, the primary source of DAGs at the plasma membrane are the phosphatidylinositol- specific phospholipase C (PI-PLC) catalyzed hydrolytic product of phosphatidylinositol-4,5 bisphosphate [PtdIns(4,5)- P 2 ]. 10,11 The DAGs are also produced at the internal membranes by a concerted action of phospholipase D (PLD) and phosphatidic acid phosphohydrolase on phosphatidylcho- line (PC). The cellular translocation of classical PKC enzymes to the plasma membrane is initially mediated by Ca 2+ binding through C2-domain, followed by C1-domain-DAG interac- tions in the presence of anionic phospholipids. In contrast, only DAG binding to the C1-domain in the presence of anionic phospholipids activates novel PKC enzymes. DAG binding allows PKC-C1 domain to penetrate into the cellular membrane and folding-out an N-terminal pseudosubstrate region, which allows access of a myriad substrates to the catalytic site of the PKC enzymes. 1,7,12,13 The presence of a homologous catalytic domain in all protein kinases, including PKCs, compelled researchers to look for an alternate substrate binding sites to regulate PKC-dependent cellular functions. 1,4,9,12,14 In the meantime, detailed mecha- nistic studies demonstrated that DAG-C1-domain interactions in the presence of anionic phospholipids can regulate PKC activity. The DAG-responsive proteins are considerably smaller Received: May 6, 2014 Revised: June 13, 2014 Published: June 17, 2014 Article pubs.acs.org/JPCB © 2014 American Chemical Society 7541 dx.doi.org/10.1021/jp5044305 | J. Phys. Chem. B 2014, 118, 7541-7553