Activators and Inhibitors of Protein Kinase C (PKC): Their Applications in Clinical Trials

Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinase, is classed into three subfamilies based on their structural and activation characteristics: conventional or classic PKC isozymes (cPKCs; α, βI, βII, and γ), novel or non-classic PKC isozymes (nPKCs; δ, ε, η, and θ), and atypical PKC isozymes (aPKCs; ζ, ι, and λ). PKC inhibitors and activators are used to understand PKC-mediated intracellular signaling pathways and for the diagnosis and treatment of various PKC-associated diseases, such as cancers, neurological diseases, cardiovascular diseases, and infections. Many clinical trials of PKC inhibitors in cancers showed no significant clinical benefits, meaning that there is a limitation to design a cancer therapeutic strategy targeting PKC alone. This review will focus on the activators and inhibitors of PKC and their applications in clinical trials.

PKCs are involved in multiple signal transduction systems that control cell proliferation, differentiation, survival, invasion, migration, and apoptosis. For these reasons, PKCs are regarded as important targets for the treatment of various diseases, such as cancers, neurological diseases (e.g., Alzheimer's disease (AD)), cardiovascular diseases (e.g., heart failure), and infections (e.g., acquired immunodeficiency syndrome) (for review see [2,[5][6][7]). PKC inhibitors and activators can be used for the treatment of various PKC-associated diseases. In this review, we will focus on the activators and inhibitors of PKC and their applications in clinical trials.

Structure of PKC Isozymes
Several important review articles have already been reported regarding the structure of PKC isozymes [1][2][3]. All PKC isozymes consist of a regulatory domain containing the C1 and C2 domains, a catalytic (kinase) domain containing C3 (N-terminal lobe (N-lobe) domain) and C4 domain (C-terminal lobe (C-lobe) domain), and variable regions (V1-V5) ( Figure 1). While the C1 domain of cPKCs and nPKCs interacts with diacylglycerol (DAG), the single C1 domain of aPKCs cannot bind to DAG. The C2 domain of cPKCs binds to Ca 2+ , but not the C2-like domain of nPKCs. The C3 domain contains an ATP-binding site, and the C4 domain has a substrate-binding site. Although all PKC isozymes do not contain the phosphatidylserine (PS)-binding domain, PS, either alone or with DAG and Ca 2+ , is essential for PKC activation [1][2][3].
The regulatory region of all PKC isozymes contains an autoinhibitory pseudosubstrate domain that inhibits kinase activity by interacting with the substrate binding site within the catalytic region. The catalytic domain contains three phosphorylation motifs: an activation loop, a turn motif, and a hydrophobic motif [1][2][3]. Moreover, aPKCs have a protein-protein-interacting region known as Phox and Bem 1 (PB1) at the N-terminus of the regulatory domain. The PB1 domain binds with partitioning-defective protein 6 (Par6), p62 (also known as sequestosome 1, SQSTM1), or mitogen-activated protein kinase kinase 5 (MEK5) [8,9].

PKC Inhibitors
Most of PKC inhibitors are C3 domain-binding inhibitors (ATP competitive PKC inhibitors), but C1 domain-(DAG competitive PKC inhibitors) and C4 domain-binding PKC inhibitors (substrate competitive PKC inhibitors) have also been reported. Among the C1-domain binding agents, bryostatin-1 acts as both an activator and inhibitor of PKC.

C2 Domain-Binding PKC Inhibitors (Ca 2+ Competitive PKC Inhibitors)
cPKC isozymes contain a C2 domain that binds to Ca 2+ , and the nPKC isozymes have a C2-like domain that cannot bind to Ca 2+ but binds to phosphotyrosines [19]. There are no reports regarding PKC inhibitors that can block the interaction with the C2 domain. However, PKCβ C2 region-derived peptides, such as C2-1 (KQKTKTIK), C2-2 (MDPNGLS-DPYVKL), and C2-4 (SLNPEWNET), inhibit the binding of PKCβ C2 fragment to the receptor for activated C-kinase (RACK). These peptide inhibitions specifically block the translocation and function of cPKC isozymes containing the C2 domain, but not nPKC isozymes containing the C2-like domain [20,21].

C3 Domain (N-Lobe Domain)-Binding PKC Inhibitors (ATP Competitive PKC Inhibitors)
Among PKC inhibitors, ATP competitive small molecule inhibitors have been broadly developed and applied in clinical trials. ATP competitive inhibitors interact with the ATPbinding pocket (C3 domain). Their IC 50 values depend on the affinity of inhibitor and the amount of added ATP [22]. High sequence homology and structural similarity in the C3 domain of PKC isozymes are major obstacles in the development of PKC isozyme-specific inhibitors [3].
Furthermore, Phase I/II clinical trials of UCN-01 have been performed in combination with irinotecan in patients with metastatic triple negative breast cancer (TNBC: negative for estrogen receptor, progesterone receptor, and HER2) [47,48]. Although impressive clinical activity was not obtained, a phase II study reported that effective CHK1 inhibition could enhance chemotherapy-induced apoptosis in TP53-mutant tumors [48]. Phase I/II studies of UCN-01 and topotecan were performed in patients with advanced ovarian cancer [49,50], but significant clinical benefit was not observed in the phase II study [50]. No further clinical trials of UCN-01 have been conducted after these studies.
Midostaurin (4'-N-benzoylstaurosporine): Midostaurin (also known as PKC412; CGP 41251) is a staurosporine analog isolated from Streptomyces staurosporeus. Similar to staurosporine, midostaurin is an ATP-competitive inhibitor and inhibits multiple protein kinases. Although midostaurin has lower inhibitory activity for PKC, its specificity for PKC is higher compared with staurosporine [51,52]. Midostaurin treatment inhibits the growth of various cancer cells and reverses P-glycoprotein-mediated multidrug resistance of cancer cells by interfering with P-glycoprotein function [51,52].
FMS-like tyrosine kinase 3 (FLT3) mutations with internal tandem duplication (ITD) are associated with high leukemic burden and poor prognosis in patients with acute myeloid leukemia (AML) [53]. FLT3/ITD mutations stimulate the tyrosine kinase activity of FLT3, resulting in growth factor-independent proliferation of FLT3/ITD-mutant AML cells [53,54]. G1 arrest and apoptosis were observed in midostaurin-treated FLT3-mutant leukemia cells by direct inhibition of tyrosine kinase (IC 50 ≤ 10 nM) [55]. In a recent phase III trial, the addition of midostaurin to standard chemotherapy significantly prolonged overall and event-free survival in mutant FLT3-positive AML patients [56].
Furthermore, systemic mastocytosis is a heterogeneous group of disorders caused by the abnormal accumulation of mast cells in organs, such as the bone marrow, liver, spleen, gastrointestinal tract, and skin. Most patients with systemic mastocytosis have an Asp816Val (D816V) mutation in the KIT receptor tyrosine kinase [57]. Midostaurin treatment significantly reduced the percentage of peripheral blood mast cells and serum histamine levels in patients with systemic mastocytosis through inhibition of KIT tyrosine kinase [58]. In addition, midostaurin induced apoptosis and downregulation of CD2 and CD63 [59] and inhibited IgE-dependent upregulation of CD63 in the mast cell leukemia cell line HMC-1 [60], resulting in enhanced inhibition of cell growth. A phase II trial showed significant clinical benefits in patients with advanced systemic mastocytosis after oral treatment with midostaurin [61,62]. However, no unexpected toxicity was observed with a median follow-up of 10 years after the phase II trial [61].
Midostaurin has been approved by the Food and Drug Administration (FDA) since April 2017 for the treatment of newly diagnosed adult AML patients with mutant FLT3positive or adult patients with systemic mastocytosis with associated hematological neoplasm, or mast cell leukemia (https://www.fda.gov/drugs/resources-information-approveddrugs/midostaurin) (access on 10 September 2020). Midostaurin treatment indicated higher cost-effectiveness in mutant FLT3-positive adult AML patients compared to the standard of care in these patients [63]. In a phase II hypothesis-generating trial, the addition of mi-dostaurin to intensive chemotherapy increased event-free survival at 2 years by 39% (95% confidence interval (CI), 33-47%) and 34% (95% CI, 24-47%) in younger and older patients, respectively, compared to historical controls treated within five prospective trials [64]. In addition, further clinical trials of midostaurin are in progress [65].
As a result, although midostaurin was originally developed as a PKC inhibitor, its success in clinical trials is mainly due to the inhibition of tyrosine kinase. However, it is not clear whether these midostaurin-induced positive results in AML patients are caused owing to inhibition of tyrosine kinase alone or both tyrosine kinase and PKC. Nevertheless, while it is true that midostaurin-mediated inhibition of tyrosine kinases is effective against AML, other tyrosine kinase inhibitors (e.g., gilteritinib and quizartinib) also show significantly improved clinical events in patients with FLT3-mutated AML [66,67].
Despite its inhibitory effects on cancer cells, a phase II trial of enzastaurin in combination with bevacizumab [78] and a phase III trial of enzastaurin alone [79] showed no clear clinical benefit in patients with recurrent malignant gliomas. In a phase III trial of enzastaurin, patients with high-risk diffuse large B-cell lymphoma (DLBCL) received a daily dose of enzastaurin (500 mg) orally for 3 years, but no significant improvement in disease-free survival was observed [80].
Furthermore, in a phase II trial of enzastaurin, there were no significant clinical benefits in patients, with previously treated multiple myeloma [81], with brain metastasis after whole brain radiotherapy [82], with epithelial ovarian or primary peritoneal carcinoma [83], with relapsed or refractory mantle cell lymphoma [84], with metastatic breast cancer previously treated with an anthracycline-and a taxane-containing regimen [85], and with relapsed or refractory advanced cutaneous T-cell lymphoma [86].
In addition, several phase II studies of enzastaurin in combination with other anticancer drugs have been conducted in patients with various cancers, such as erlotinib or erlotinib/ enzastaurin in patients with non-small-cell lung cancer (NSCLC) [87], temozolomide or temozolomide/enzastaurin plus radiation therapy in patients with glioblastoma multiforme and gliosarcoma [88], docetaxel/prednisone or docetaxel/prednisone/enzastaurin in patients with castration-resistant metastatic prostate cancer [89], paclitaxel/carboplatin or paclitaxel/carboplatin/enzastaurin in patients with advanced ovarian cancer [90], 5fluorouracil/leucovorin plus bevacizumab with or without enzastaurin in patients with metastatic colorectal cancer [91], pemetrexed or pemetrexed/enzastaurin in patients with advanced NSCLC [92], and gemcitabine or gemcitabine/enzastaurin in patients with advanced or metastatic pancreatic cancer [93]. However, these phase II trials failed to show any clinical benefits (e.g., progression-free survival) in these combinatorial treatments.
A phase I trial has also been conducted in children with recurrent central nervous system malignancies [94]. Despite the absence of objective responses, enzastaurin was well tolerated in children and the recommended phase II dose is 440 mg/m 2 /day administered once, daily [94].
PKCβ is highly expressed in the retina. Ruboxistaurin reduces the pathogenesis of diabetic retinopathy in diabetic rats by inhibiting PKCβ and hence preventing the increase in leukostasis and decrease in retinal blood flow [95,96]. In addition, it reduced the expression of endothelin-1 and platelet-derived growth factor in the retina [97] and inhibited vascular endothelial growth factor-induced phosphorylation of Akt and extracellular signal-regulated kinase 1/2 [98].
Patients with diabetic nephropathy exhibit either a painless syndrome with loss of sensation or a painful disorder accompanied by hyperalgesia and allodynia [99,100]. Ruboxistaurin attenuates diabetic hyperalgesia in diabetic rats by reducing the neuronal nitric oxide synthase-cGMP system [101]. Ruboxistaurin also inhibits NADPH oxidasemediated production of reactive oxygen species in the kidney of diabetic rats, which is associated with renal injury [102]. Ruboxistaurin (10 µM) binds to the ATP binding site of 3phosphoinositide dependent protein kinase-1 (PDK1), which is involved in the insulin-like growth factor signaling pathway, and exhibits higher inhibitory effects on PDK1, compared to other bisindolylmaleimides (each 10 µM), such as Bis-1, -2, -3, and -8 [73].
Transforming growth factor-β (TGF-β) activation stimulates the phosphoinositide-3kinase/Akt pathway that accelerates renal injury and dysfunction [103]. Ruboxistaurin treatment reduces high glucose-induced Akt and TGF-β activation in mesangial cells and Akt activation in the renal cortex of diabetic rats [104]. In addition, ruboxistaurin-treated rat models of diabetic nephropathy showed a significant decrease in osteopontin expression, in addition to macrophage infiltration, interstitial fibrosis, and TGF-β activity in tubular epithelial cells of the cortex [105]. Based on these results, ruboxistaurin has been considered as a potential therapeutic agent for diabetic nephropathy and retinopathy.
A phase III study investigated the effect of ruboxistaurin (32 mg/day) on vision loss in patients with moderate to severe non-proliferative diabetic retinopathy. Reduced occurrence of sustained moderate visual loss (≥15-letter decline in visual acuity sustained for the last 6 months of study participation) was observed in patients with greatest ruboxistaurin exposure (~5 years), compared to control patients (~2-year ruboxistaurin exposure) [106]. Furthermore, two phase III trials of ruboxistaurin have been conducted in patients with (Early Treatment Diabetic Retinopathy Study) retinopathy level 20 to 47D or 35B to 53E, and no prior panretinal or focal photocoagulation in at least one eye at baseline. Although ruboxistaurin treatment showed an approximately 50% reduction in sustained moderate vision loss, caused due to diabetic macular edema, statistical significance was not achieved [107]. For patients with diabetes and symptomatic diabetic peripheral neuropathy, two identical, phase III, parallel, randomized, double-blind, placebo-controlled trials of ruboxistaurin (32 mg/day) have been performed, but these trials failed to show a significant and progressive improvement in symptoms [108]. Based on these findings, ruboxistaurin has not been used for further clinical trials.
pyrrole-2,5-dione} is a potent and selective pan-PKC inhibitor, with various K i values for PKC isozymes, such as 0.95 nM for PKCα, 0.64 nM for PKCβ, 0.22 nM for PKCθ, and 1.8-3.2 mM for PKCδ, ε, and η [109,110]. Sotrastaurin exhibits immunosuppressive functions, such as inhibition of T-cell activation [111] and suppression of B-cell antibody response [112]. Sotrastaurin has been reported to prevent T-cell-mediated rejection in liver and kidney transplantation [113]. The efficacy and safety of sotrastaurin alone in de novo kidney transplant recipients [114], sotrastaurin plus tacrolimus in de novo liver [115], and kidney transplant recipients [116], and sotrastaurin plus everolimus in de novo kidney transplant recipients [117] were evaluated through phase II clinical trials. All these clinical trials exhibited adverse effects and high failure rates with respect to efficacy.
Furthermore, sotrastaurin showed growth inhibitory effects on CD79-mutant DLBCL through NF-κB pathway inhibition and induction of G 1 -phase cell-cycle arrest and/or cell death [118,119]. However, a phase Ib study of safety and efficacy of sotrastaurin and everolimus (mTOR inhibitor) in patients with CD79-mutant or activated B-cell-like subtype DLBCL exhibited suboptimal tolerability of the combination treatment, resulting in no implementation of phase II (NCT01854606).
In addition, a recent phase I study of sotrastaurin in patients with metastatic uveal melanoma showed that it was well tolerated, and modest clinical activity was observed, with a low objective response rate (3%) [120].

Peptide Inhibitors
PKC peptide inhibitors are mainly divided into (1) peptides derived from PKC protein fragment and (2) peptides obtained by the mutation of phosphorylation sites of PKC substrates. Myristoylated (myr-PKC) inhibitors show higher inhibitory effects on target PKC isozymes than non-myristoylated PKC peptide inhibitors [128][129][130][131]. Moreover, D-type amino acids are used to increase the inhibitory efficiency of peptide inhibitors [131,132]. Furthermore, peptide length can influence the potency of peptide inhibitor. Reduction in peptide length leads to decreased potency of the inhibitor [130,133].
In a phase II clinical trial of PKCδ V1 region-derived peptide (also known as delcasertib or KAI-9803) [139], its intravenous injection into patients within 6 h of undergoing primary percutaneous coronary intervention for acute ST elevation myocardial infarction did not improve clinical events and left ventricular function and did not reduce expression of biomarkers of myocardial injury [140]. Furthermore, in a phase II clinical trial of PKCε V1 region-derived peptides (KAI-1678) [136], its subcutaneous injection, for the treatment of neuropathic pain, in patients with postherpetic neuralgia failed to show a significant reduction in pain intensity ( [141]. Mutant peptide inhibitors: Mutant peptide inhibitors are generated by replacing the phosphorylation sites (Ser or Thr) with mostly Ala [130,142]. However, a study has reported that Cys replacement instead of Ala increases the potency of the inhibitor [129]. Mutant peptide inhibitors block the binding of the substrate to PKC. However, these mutant peptide inhibitors show very low inhibitory efficiencies for PKC [130].

Other Inhibitors Binding to the C4 Domain
Chelerythrine (IC 50 = 0.66 µM), a natural benzophenanthridine alkaloid isolated from Chelidonium majus, is a competitive inhibitor with respect to the phosphate acceptor (histone IIIS) and a noncompetitive inhibitor with respect to ATP, meaning that it binds to the C4 domain of the PKC catalytic region [143]. It has broad biological activities, such as anticancer [144], anti-inflammatory [145], antiviral [146], antifungal [147], and antibacterial effects [148]. Chelerythrine inhibits the growth of cells in various ranges of IC 50 values.  [151]. On the other hand, a study suggested that, while chelerythrine could not inhibit PKC activity, it could stimulate PKC activity in the cytosolic fractions of rat and mouse brain tissues at concentrations of up to 100 µM [152].
Riluzole binds to the catalytic domain of PKC, but ATP concentrations do not affect riluzole-mediated PKC inhibition. This means that riluzole is not a competitive inhibitor of ATP and binds to the C4 domain [153]. Riluzole is an FDA-approved medication that has neuroprotective properties and is used to treat amyotrophic lateral sclerosis. PKC is activated in amyotrophic lateral sclerosis, and riluzole-mediated PKC inhibition may be involved in the neuroprotective mechanism [153,154]. Furthermore, riluzole (0.1-10 µM) inhibits VEGF-stimulated PKC βII activation and cell proliferation in bovine retinal endothelial cell and human umbilical vein endothelial cell cultures [155]. Riluzole (30 µM) also inhibits PKC activity in the membrane of cortical cells [153].

Atypical PKC Inhibitors
There are few reports on PKC isozyme-specific inhibitors, but some aPKC-specific inhibitors have recently been reported and are summarized below. These inhibitors block the activity of aPKC by binding to either the PB domain that exists at the N-terminus of aPKC or to the catalytic domain ( Figure 1). The therapeutic efficacy of aPKC inhibitors in patients is yet to be investigated in clinical trials.

ZIP (PB Domain)
While ZIP (SIYRRGARRWRKL) shows high binding affinity for aPKC, it can also bind to multiple PKC isozymes [135]. Its potential inhibitory activity for PKCι and PKCζ is nearly equal (K i (95% CI) = 1.43 and 1.7 µM, respectively) [159]. ZIP does not inhibit the catalytic activity of the kinase domain of PKCζ but prevents the interaction of the PB1 domain of PKCζ with that of p62 by binding to an acidic surface on the PB1 domain of p62 [160,161].
PKMζ, an N-terminal truncated isoform of PKCζ, plays a critical role in the maintenance of long-term potentiation, long-term memory, and chronic pain [159,[162][163][164]. ZIP is a candidate inhibitor for PKMζ. Despite its dependence on substrate and kinase concentrations, myr-ZIP completely inhibits PKMζ activity in the range of 5-10 µM and its IC 50 value for PKMζ ranges from 0.076 to 2 µM [135,159,163,165]. On the other hand, ZIP-induced excitotoxic death of cultured neurons at 5-10 µM has been reported [166]. In animal disease models, ZIP administration alleviated or prevented pain-related disorders, such as chronic visceral pain [167] and neuropathic pain [168], and memory-related disorders, such as anxiety in autism [169] and fear-mediated anxiety [170,171]. However, there are no data on the application of ZIP inhibitor peptide in clinical trials.
The phase I study of aurothiomalate has been conducted in patients with PKCιoverexpressed cancers, such as advanced NSCLC, ovarian cancer, and pancreatic cancer [180]. A feasibility study for enrolling asymptomatic ovarian cancer patients with increased levels of CA-125 (10 patients) has also been carried out by oral administration of auranofin, which resulted in decreased levels of CA-125 in one patient [181]. Furthermore, a phase I/II clinical trial of auranofin (NCT01419691) has been conducted in patients with chronic lymphocytic leukemia, small lymphocytic and prolymphocytic lymphoma [182].

Bryostatin-1
Bryostatin-1, a macrocyclic lactone isolated from a marine invertebrate, binds to the C1 domain of PKC and acts both as an activator and inhibitor for PKC. For example, short-term exposure to bryostatin-1 stimulates PKC activation, while long-term exposure promotes downregulation of PKC activity [202].

Bryostatin-1 as a PKC Inhibitor
Bryostatin-1 competes with cancer-promoting PKC ligands (e.g., DAG and phorbol esters) to bind to PKC since it has the same binding site (C1 domain) as PKC ligands. Based on these functions, phase II studies of bryostatin-1 with other anticancer drugs have been performed in patients with various cancers. Phase II trials of single-agent bryostatin-1 showed no clinical effects in several cancers, such as metastatic malignant melanoma [203][204][205], metastatic renal cell carcinoma [206,207], metastatic colorectal cancer [208], NHL [209], relapsed multiple myeloma [210], advanced sarcoma and advanced head and neck cancer [211], metastatic or recurrent squamous cell carcinoma of the head and neck [212], squamous cell carcinoma of the cervix [213], and recurrent epithelial ovarian carcinoma [214].
Furthermore, no clinical responses were observed in phase II studies of bryostatin-1/paclitaxel in patients with advanced pancreatic carcinoma [215], advanced NSCLC [216], and advanced or recurrent carcinoma of the cervix [217], as well as in a phase II study of four different doses of bryostatin-1/interleukin-2 treatment in patients with renal cell carcinoma [218].
On the other hand, bryostatin-1/paclitaxel treatment in a phase II study resulted in a superior response rate in patients with untreated, advanced gastric or gastroesophageal junction adenocarcinoma, compared to paclitaxel alone [219]. Another phase II study of the same treatment in patients with advanced esophageal cancer, despite potential anti-tumor activity, was prematurely closed because of excessive toxicity [220].
In patients with recurrent platinum-sensitive or resistant ovarian cancer, a phase II trial of bryostatin-1/cisplatin showed a modest response rate. However, it increased toxicity in platinum-pretreated patients [221]. A phase II study of bryostatin-1/vincristine showed efficacy in select patients (overall response rate of 31%) with aggressive B-cell NHL which relapsed after autologous stem cell transplantation [222].

Bryostatin-1 as a PKC Activator
Previous studies have suggested that bryostatin-1- [223,224] or PMA- [225] mediated PKCε activation could reduce amyloid-β levels and prevent learning and memory deficits in mice with AD. In a single-dose (25 µg/m 2 ) phase IIa clinical trial, bryostatin-1 administration to patients with AD showed cognitive improvement in the first 24 weeks through elevated PKCε levels [226]. A recent phase II study of bryostatin-1 (20 µg) in patients with AD suggested that the primary endpoint at 13 weeks showed no significance for the full analysis set (FAS), but the improved signals of Severe Impairment Battery (SIB) scores were obtained at 13 weeks for the Completers Set and for both data sets (FAS + SIB) at 15 weeks, compared to those of placebo patients [227].
Furthermore, several studies showed that bryostatin-1-mediated PKC activation could reactivate latent (inactive) HIV-1 [228,229]. However, a phase I clinical trial of bryostatin-1 (20 µg/m 2 ) in HIV-1infected patients exhibited no effect on PKC activity or on the transcription of latent HIV-1. These negative results may be due to low plasma concentrations of bryostatin-1 [230].

Perspectives for Research and Application of PKC Inhibitors and Activators
PKCs are regarded as attractive targets for cancer therapy because their hyperactivation in many cancers [76,141,179,196]. For these reasons, clinical trials of PKC inhibitors have focused on the treatment of many cancers. However, PKCs are also associated with various diseases, such as neurological diseases, cardiovascular diseases, and infection. Activators and inhibitors of PKC can be used for the treatment of these diseases.
PKC and AD: As briefly mentioned above, recently, PKC activation has been attracting attention as a novel therapeutic strategy for AD. For example, reduced PKCε levels, but increased β-amyloid (Aβ) levels, were found in the hippocampus and temporal pole areas of patients with AD [231]. PKCε promotes the expression of brain-derived neurotrophic factor (BDNF) in the brain, which plays a role in the growth and maintenance of neuronal networks. However, reduced expression of PKCε and BDNF has been observed in the hippocampal neuron in patients with AD [224]. In fact, a phase IIa clinical trial showed that bryostatin-1-mediated activation of PKCε could result in cognitive improvement in the first 24 weeks [226]. In addition, PKCα activation can be a useful tool for treating AD [232].
These results suggest that the biological function of PKC isozyme in AD progression could be different, and that PKC might be a therapeutic potential target for AD.
PKC and HIV: Recently, PKC activators have received attention as latency-reversing agents in HIV treatment. They reactivate latent HIV-1 within immune cells (e.g., CD4 and CD8 T cells) through activation of the NF-κB transcription factor pathway and enhance the recognition and removal of HIV by the immune system [236,237]. Despite the failure of a phase I clinical trial using the PKC activator bryostatin-1, which could be due to its low plasma concentrations [230], PKC activators are still regarded as one of the most promising agents for reversing HIV-1 latency. Therefore, PKC activators may remarkably increase therapeutic efficacy of HIV in combination with antiretroviral drugs [238,239].
PKC, cardiac disease, and heart failure: PKCs are good therapeutic targets for the treatment of cardiac disease and heart failure. For example, PKCα, PKCβ, PKCδ, and PKCε are targeted for treating cardiac hypertrophy, PKCβ, PKCδ, and PKCε are targeted for treating heart failure, and PKCθ for lowing heart transplant rejection [6,240,241]. However, there are no reports regarding clinical trials of PKC activators or inhibitors for cardiac disease and heart failure, except phase I/II trials of delcasertib in patients with myocardial infarction [140,242].

Summary and Overall Conclusions
Activators and inhibitors of PKC and their applications in clinical trials are summarized in Table 1. Despite many clinical trials of PKC inhibitors in cancers, most of them showed no significant clinical benefits. On the other hand, a phase III trial of midostaurin plus standard chemotherapy in mutant FLT3-positive AML patients [56] and a phase II trial of midostaurin alone in patients with advanced systemic mastocytosis [61] exhibited significant clinical benefits, such as enhanced overall response rate, prolonged event-free survival, and low unexpected toxicity. These clinical benefits of midostaurin are mainly due to the inhibition of tyrosine kinase, but not PKC, as mentioned above. Further studies are needed to investigate whether these midostaurin-induced clinical benefits are caused by inhibition of tyrosine kinase alone or both tyrosine kinase and PKC. However, PKC inhibitors may increase clinical efficacy in combination with tyrosine kinase inhibitors. The reason why PKC-targeted inhibitors show no significant clinical benefits in several clinical trials of cancers is not clear yet. However, we speculate that the following three possibilities might contribute to little or no clinical benefits in clinical trials of PKC inhibitors: (1) Isozyme-nonspecific PKC inhibitors. PKC isozymes are involved in multiple biological functions in cancer cells, such as tumorigenic or anti-tumorigenic, pro-apoptotic or anti-apoptotic, and pro-proliferative or anti-proliferative [243,244]. Isozyme-nonspecific PKC inhibitors, especially, ATP competitive PKC inhibitors, can block the activation of PKC isozymes with both tumorigenic, anti-apoptotic, and pro-proliferative function as well as anti-tumorigenic, pro-apoptotic, and anti-proliferative function. Therefore, the use of isozyme-nonspecific PKC inhibitors as chemotherapy drugs could lead to decreased therapeutic efficacy in cancers.
(2) PKC mutations. PKC loss-of-function mutations are found in a multitude of cancers [2,244]. This suggests that PKC inhibitors fail to exhibit significant clinical benefits in patients with PKC-mutated cancers. However, there are no reports that show whether PKC mutations in cancers have been investigated in clinical trials of PKC inhibitors.
(3) Limitation of PKC as therapeutic target. PKCs related to pro-proliferative and anti-apoptotic function are significantly activated in cancers. Although the activation of these PKCs is inhibited by PKC inhibitors, other cellular signals (e.g., AKT) that are pro-proliferative and anti-apoptotic in cancer may be substituted for PKCs. On the other hand, certain inhibitors of PI3Ks that are upstream of PKCs and AKT show significant clinical benefits in cancer treatment [245][246][247].
Therefore, so long as cellular signals that have similar functions as PKCs are activated in cancers, the inhibition of PKC alone may result in little or no clinical benefits in clinical trials. In fact, accumulating evidence suggests that there is a limitation to design a cancer therapeutic strategy targeting PKC alone (Table 1)