Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain–targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.

Prostate cancer is one of the most common cancers in men. Although it has a relatively high 5‐year survival rate, development of resistance to standard androgen‐deprivation therapy is a significant clinical problem. Therefore, novel therapeutic strategies are urgently needed. The protein kinase C ( PKC ) family is a putative prostate cancer drug target, but so far no PKC ‐targeting drugs are available for clinical use. By contrast to the standard approach of developing PKC inhibitors, we have developed isophthalate derivatives as PKC agonists. In this study, we have characterized the effects of the most potent isophthalate, 5‐(hydroxymethyl)isophthalate 1a3 ( HMI ‐1a3), on three prostate cancer cell lines ( LNC aP, DU 145, and PC 3) using both 2D and 3D cell culture models. In 2D cell culture, HMI ‐1a3 reduced cell viability or proliferation in all cell lines as determined by the metabolic activity of the cells (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide assay) and thymidine incorporation. However, the mechanism of action in LNC aP cells was different to that in DU 145 or PC 3 cells. In LNC aP cells, HMI ‐1a3 induced a PKC ‐dependent activation of caspase 3/7, indicating an apoptotic response, whereas in DU 145 and PC 3 cells, it induced senescence, which was independent of PKC . This was observed as typical senescent morphology, increased β‐galactosidase activity, and upregulation of the senescence marker p21 and downregulation of E2F transcription factor 1. Using a multicellular spheroid model, we further showed that HMI ‐1a3 affects the growth of LNC aP and DU 145 cells in a 3D culture, emphasizing its potential as a lead compound for cancer drug development.

Abnormal protein kinase C (PKC) function contributes to many pathophysiological processes relevant for Alzheimer's disease (AD), such as amyloid precursor protein (APP) processing. Phorbol esters and other PKC activators have been demonstrated to enhance the secretion of soluble APPα (sAPPα), reduce the levels of β-amyloid (Aβ), induce synaptogenesis, and promote neuroprotection. We have previously described isophthalate derivatives as a structurally simple family of PKC activators. Here, we characterised the effects of isophthalate derivatives HMI-1a3 and HMI-1b11 on neuronal viability, neuroinflammatory response, processing of APP and dendritic spine density and morphology in in vitro. HMI-1a3 increased the viability of embryonic primary cortical neurons and decreased the production of the pro-inflammatory mediator TNFα, but not that of nitric oxide, in mouse neuron-BV2 microglia co-cultures upon LPS- and IFN-γ-induced neuroinflammation. Furthermore, both HMI-1a3 and HMI-1b11 increased the levels of sAPPα relative to total sAPP and the ratio of Aβ42/Aβ40 in human SH-SY5Y neuroblastoma cells. Finally, bryostatin-1, but not HMI-1a3, increased the number of mushroom spines in proportion to total spine density in mature mouse hippocampal neuron cultures. These results suggest that the PKC activator HMI-1a3 exerts neuroprotective functions in the in vitro models relevant for AD by reducing the production of TNFα and increasing the secretion of neuroprotective sAPPα.

Targeting cytotoxic 4β-phorbol esters toward cancer tissue was attempted by conjugating a 4β-pborbol derivative with substrates for the proteases prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) expressed in cancer tissue. The hydrophilic peptide moiety was hypothesized to prevent penetration of the prodrugs into cells and prevent interaction with PKC. Cleavage of the peptide in cancer tumors was envisioned to release lipophilic cytotoxins, which subsequently penetrate into cancer cells. The 4β-phorbol esters were prepared from 4β-phorbol isolated from Croton tiglium seeds, while the peptides were prepared by solid-phase synthesis. Cellular assays revealed activation of PKC by the prodrugs and efficient killing of both peptidase positive as well as peptidase negative cells. Consequently no selectivity for enzyme expressing cells was found. KEYWORDS: 4β-Phorbol ester, protease-assisted targeting, targeted chemotherapy, prodrug, prostate-specific antigen. prostate-specific membrane antigen P rostate cancer (PCa) is a major cause of death by cancer in men in high-income countries. 1 In the initial stage, PCa mainly consists of cells that are androgen-dependent, and the growth can be retarded by hormone therapy. 2 Unfortunately, in later stages hormone refractory cells dominate (castrationresistant prostate cancer, CRPC). 2,3 At this stage the use of common chemotherapeutics is complicated by the slow proliferation of the cancer tissue, since chemotherapeutics like taxanes, doxorubicine, or vincristine target the proliferative stages of cancer. Thus, selectivity is obtained by the faster division rate for cancer cells. 3−6 Therefore, an urgent need for drugs against late-stage PCa exists.Preclinical evidence supports the idea that drugs targeting protein kinase C (PKC) may be useful in treatment of CRPC. 7 The PKC family comprises ten serine/threonine kinases, which can be divided into three groups: (i) conventional PKC (cPKCs: -α, -βI, -βII, and -γ), (ii) novel PKCs (nPKCs: -δ, -ε, -θ, and -η), and (iii) atypical PKCs (aPKCs: -ζ, -ι, and λ). Expression and function of different PKC isoforms are context-

Colorectal cancer is the third most commonly occurring cancer in men and the second in women. The global burden of colorectal cancer is projected to increase to over 2 million new cases with over 1 million deaths within the next 10 years and there is a great need for new compounds with novel mechanisms of action. Our group has developed PKC modulating isophthalic acid derivatives that induce cytotoxicity towards human cervical and prostate cancer cell lines. In this study, we investigated the effects of 5-(hydroxymethyl)isophthalate 1a3 (HMI-1a3) on colorectal cancer cell lines (Caco2, Colo205 and HT29). HMI-1a3 inhibited cell proliferation, decreased cell viability and induced an apoptotic response in all studied cell lines. These effects, however, were independent of PKC. Using serine/threonine kinome profiling and pharmacological kinase inhibitors we identified activation of the cAMP/PKA pathway as a new mechanism-of-action for HMI-1a3-induced anti-cancer activity in colorectal cancer cell lines. Our current results strengthen the hypothesis for HMI-1a3 as a potential anti-cancer agent against various malignancies.