The DAG-responsive C1 domain as a drug target: structure-activity and in vitro pharmacology of isophthalate derivatives

Diacylglycerol (DAG) is a ubiquitous lipid second messenger that transmits signals from cell membrane receptors to intracellular effector proteins containing a specialized DAG recognition motif, the C1 domain. The protein kinase C (PKC) family of serine/threonine kinases is the bestcharacterized member of DAG effectors, but there are also six other families of proteins with a DAGresponsive C1 domain: (1) the protein kinase D (PKD) family; (2) the myotonic dystrophy kinaserelated Cdc42-binding kinases (MRCKs); (3) the DAG kinases (DGKs); (4) the Munc13 proteins; (5) the chimaerins; and (6) the Ras guanyl-releasing proteins (RasGRPs). DAG-mediated signalling regulates many cellular functions such as cell proliferation, survival, differentiation, apoptosis and motility − processes that are often deregulated in cancer. As key mediators of these processes, several of the DAG effectors are regarded as promising targets for cancer drug development. Furthermore, PKC contributes to neuronal plasticity and inhibits many pathophysiological processes related to Alzheimer’s disease. Activation of PKC is therefore considered a potential future therapeutic strategy for the treatment of Alzheimer’s disease. The C1 domain thus represents a well-recognized drug target. However, the existing C1 domain ligands are extremely complex in their chemical structure and from the drug development point of view new C1 domain ligands with a feasible synthesis route are needed. The purpose of these studies was to characterize the structure-activity relationships (SAR) of a novel group of C1 domain ligands, derivatives of 5-(hydroxymethyl)isophthalic acid. These compounds have a simple chemical structure and they are easy to synthesize. Furthermore, these studies aimed to investigate the in vitro pharmacology of the isophthalate derivatives especially focusing on their effects on cell proliferation and morphology. The SAR studies revealed the structural elements indispensable for binding to the C1 domain of PKC: the hydroxymethyl group, both of the ester groups and sufficiently hydrophobic ester substituents. Importantly, the SAR model also held true with β2-chimaerin, another C1-domain containing DAG effector. The active isophthalates bound to PKC with low micromolar affinities, and a selected example of these, HMI-1a3, was also shown to bind to the C1 domains of PKD1 and MRCKα at similar concentrations. Several isophthalates had an antiproliferative effect in HeLa human cervical carcinoma cells. HMI-1a3 exhibited the most potent cytotoxic and antiproliferative effect of the derivatives tested. The isophthalates with no affinity to the C1 domain (e.g. NI-15e, the inactive derivative of HMI-1a3) had no effect on HeLa cell viability or proliferation, suggesting a C1 domain-mediated effect. HMI-1a3 also induced a morphological change characterized by cell elongation. It was accompanied with a marked reorganization of actin cytoskeleton: loss of focal adhesions and actin stress fibres. The elongation-inducing effect of HMI-1a3 was inhibited by an MRCK inhibitor, and thus seems to be at least partially mediated by MRCK. However, the mechanism of its antiproliferative effect remains unclear and may be mediated by several of the DAG effectors. In SH-SY5Y neuroblastoma cells, which are widely used as an in vitro model for neuronal differentiation, HMI-1b11 inhibited cell proliferation and supported neurite growth. HMI-1a3 induced SH-SY5Y cell differentiation as well, but unlike HMI-1b11 it also induced cell death. Again, derivatives with poor binding affinity to the C1 domain had no effect. The HMI-1b11-induced response was accompanied by PKC-dependent ERK1/2 phosphorylation and up-regulation of GAP43, which is known to mediate neuronal differentiation and to contribute to neurite outgrowth. In conclusion, these studies identify derivatives of 5-(hydroxymethyl)isophthalic acid as a promising novel group of C1 domain ligands. The antiproliferative and cytotoxic HMI-1a3 serves as a potential lead molecule for cancer-related drug discovery. HMI-1b11 on the other hand may be useful in drug development related to neurodegenerative diseases because of its non-toxic and differentiation-inducing properties in SH-SY5Y cells. More studies using cell-based and in vivo models are, however, needed to fully assess the potential of isophthalates in drug development.