Peptidomimetic inhibitors of the VEGF-A165/NRP-1 complex obtained by modification of the C-terminal arginine.

Inhibitors of the interaction between Neuropilin-1 (NRP-1) and Vascular Endothelial Growth Factor-A165 (VEGF-A165) hold significant promise as therapeutic and diagnostic agents directed against cancers overexpressing NRP-1. In our efforts in this field, a few series of strong and fairly stable peptide-like inhibitors of the general formula Lys(Har)1-Xaa2-Xaa3-Arg4 have been previously discovered. In the current work, we focused on Lys(Har)-Dap/Dab-Pro-Arg sequence. The aim was to examine whether replacing C-terminal Arg with its homologs and mimetics would yield more stable yet still potent inhibitors. Upon considering the results of modelling and other factors, ten novel analogues with Xaa4 = homoarginine (Har), 2-amino-4-guanidino-butyric acid (Agb), 2-amino-3-guanidino-propionic acid (Agp), citrulline (Cit), 4-aminomethyl-phenylalanine [Phe(4-CH2-NH2)] were designed, synthesized and evaluated. Two of the proposed modifications resulted in inhibitors with activity slightly lower [e.g. IC50 = 14.3 µM for Lys(Har)-Dab-Pro-Har and IC50 = 19.8 µM for Lys(Har)-Dab-Pro-Phe(4-CH2-NH2)] than the parent compounds [e.g. IC50 = 4.7 µM for Lys(Har)-Dab-Pro-Arg]. What was a surprise to us, the proteolytic stability depended more on position two of the sequence than on position four. The Dab2-analogues exhibited half-life times beyond 60 h. Our results build up the knowledge on the structural requirements that effective VEGF-A165/NRP-1 inhibitors should fulfil.

Applications of biaryl cyclization in the synthesis of cyclic enkephalin analogs with a highly restricted flexibility.

A series of 10 cyclic, biaryl analogs of enkephalin, with Tyr or Phe residues at positions 1 and 4, were synthesized according to the Miyaura borylation and Suzuki coupling methodology. Biaryl bridges formed by side chains of the two aromatic amino acid residues are of the meta-meta, meta-para, para-meta, and para-para configuration. Conformational properties of the peptides were studied by CD and NMR. CD studies allowed only to compare conformations of individual peptides while NMR investigations followed by XPLOR calculations provided detailed information on their conformation. Reliability of the XPLOR calculations was confirmed by quantum chemical ones performed for one of the analogs. No intramolecular hydrogen bonds were found in all the peptides. They are folded and adopt the type IV ß-turn conformation. Due to a large steric strain, the aromatic carbon atoms forming the biaryl bond are distinctly pyramidalized. Seven of the peptides were tested in vitro for their affinity for the µ-opioid receptor.

Structure-activity relationship studies and biological properties evaluation of peptidic NRP-1 ligands: Investigation of N-terminal cysteine importance.

Neuropilin-1 (NRP-1) is a major co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2). It may also stimulate tumour growth and metastasis independently of VEGF-A165. These functions make VEGF-A165/NRP-1 complex formation and its inhibition of great interest, where NRP-1 is the target for which effective ligands are sought. Design of peptide-like inhibitors represent a strategy with great potential in the treatment of NRP-1-related disorders. Here, we present the synthesis, molecular modelling, structure-activity relationship studies as well as biological evaluation of peptides with the branched sequences H2N-X-Lys(hArg)-Dab-Oic-Arg-OH and H2N-Lys(X-hArg)-Dab-Oic-Arg-OH. Two of the designed peptides, in which Cys was inserted in X position, expressed high affinity (∼40 nM value) for NRP-1 and were resistant to enzymatic digestion in human serum. Moreover, peptide/NRP-1 complex promoted fast intracytoplasmic protein trafficking towards the plasma membrane in breast cancer cells. Our results suggest that these compounds might be good candidates for further development of VEGF-A165/NRP-1 inhibitors.

The influence of a synthetic growth hormone-releasing hormone analogue G11 and opioid peptide biphalin on selected fibroblasts parameters relevant to wound healing.

The treatment of hard-to-heal chronic wounds is still a major medical problem and an economic and social burden. In this work, we examine the proregenerative potential of two peptides, G11 (a trypsin-resistant analogue of growth hormone-releasing hormone [GHRH]) and biphalin (opioid peptide), and their combination in vitro on human fibroblasts (BJ). G11, biphalin and their combination exhibited no toxicity against BJ cells. On the contrary, these treatments significantly stimulated proliferation and migration of fibroblasts. Under inflammatory conditions (LPS-induced BJ cells), we noticed that the tested peptides decreased the levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and interleukin 1ß (IL-1ß). This was correlated with diminished phosphorylation levels of p38 kinase, but not those of ERK1/2. We found also that G11, biphalin and their combination activated the ERK1/2 signalling pathway, which has been previously implicated in promigratory activity of some regeneration enhancers, including opioids or GHRH analogues. Potential application of their combination requires further work, in particular in vivo experiments, in which the organism-level relevance of the discussed cell-level effects would be proven and, additionally, analgesic action of the opioid ingredient could be quantified.

Novel NK1R-Targeted 68Ga-/177Lu-Radioconjugates with Potential Application against Glioblastoma Multiforme: Preliminary Exploration of Structure-Activity Relationships.

Locoregionally administered, NK1 receptor (NK1R) targeted radionuclide therapy is a promising strategy for the treatment of glioblastoma multiforme. So far, the radiopharmaceuticals used in this approach have been based on the endogenous agonist of NK1R, Substance P or on its close analogues. Herein, we used a well-known, small molecular NK1R antagonist, L732,138, as the basis for the radiopharmaceutical vector. First, 14 analogues of this compound were evaluated to check whether extending the parent structure with linkers of different lengths would not deteriorate the NK1R binding. The tested analogues had affinity similar to or better than the parent compound, and none of the linkers had a negative impact on the binding. Next, five DOTA conjugates were synthesized and used for labelling with 68Ga and 177Lu. The obtained radioconjugates turned out to be fairly lipophilic but showed rather limited stability in human plasma. Evaluation of the receptor affinity of the (radio)conjugates showed that neither the chelator nor the metal negatively impacts the NK1R binding. The 177Lu-radioconjugates exhibited the binding characteristics towards NK1R similar or better than that of the 177Lu-labelled derivative of Substance P, which is in current clinical use. The experimental results presented herein, along with their structural rationalization provided by modelling, give insight for the further molecular design of small molecular NK1R-targeting vectors.

Bifunctional Opioid/Melanocortin Peptidomimetics for Use in Neuropathic Pain: Variation in the Type and Length of the Linker Connecting the Two Pharmacophores.

Based on the mechanism of neuropathic pain induction, a new type of bifunctional hybrid peptidomimetics was obtained for potential use in this type of pain. Hybrids consist of two types of pharmacophores that are connected by different types of linkers. The first pharmacophore is an opioid agonist, and the second pharmacophore is an antagonist of the pronociceptive system, i.e., an antagonist of the melanocortin-4 receptor. The results of tests in acute and neuropathic pain models of the obtained compounds have shown that the type of linker used to connect pharmacophores had an effect on antinociceptive activity. Peptidomimetics containing longer flexible linkers were very effective at low doses in the neuropathic pain model. To elucidate the effect of linker lengths, two hybrids showing very high activity and two hybrids with lower activity were further tested for affinity for opioid (mu, delta) and melanocortin-4 receptors. Their complexes with the target receptors were also studied by molecular modelling. Our results do not show a simple relationship between linker length and affinity for particular receptor types but suggest that activity in neuropathic pain is related to a proper balance of receptor affinity rather than maximum binding to any or all of the target receptors.

Biphalin-A Potent Opioid Agonist-As a Panacea for Opioid System-Dependent Pathophysiological Diseases?

Biphalin, one of the opioid agonists, is a dimeric analog of enkephalin with a high affinity for opioid receptors. Opioid receptors are widespread in the central nervous system and in peripheral neuronal and non-neuronal tissues. Hence, these receptors and their agonists, which play an important role in pain blocking, may also be involved in the regulation of other physiological functions. Biphalin was designed and synthesized in 1982 by Lipkowski as an analgesic peptide. Extensive further research in various laboratories on the antinociceptive effects of biphalin has shown its excellent properties. It has been demonstrated that biphalin exhibits an analgesic effect in acute, neuropathic, and chronic animal pain models, and is 1000 times more potent than morphine when administered intrathecally. In the course of the broad conducted research devoted primarily to the antinociceptive effect of this compound, it has been found that biphalin may also potentially participate in the regulation of other opioid system-dependent functions. Nearly 40 years of research on the properties of biphalin have shown that it may play a beneficial role as an antiviral, antiproliferative, anti-inflammatory, and neuroprotective agent, and may also affect many physiological functions. This integral review analyzes the literature on the multidirectional biological effects of biphalin and its potential in the treatment of many opioid system-dependent pathophysiological diseases.

A Phage Display-Identified Short Peptide Capable of Hydrolyzing Calcium Pyrophosphate Crystals-The Etiological Factor of Chondrocalcinosis.

Chondrocalcinosis is a metabolic disease caused by the presence of calcium pyrophosphate dihydrate crystals in the synovial fluid. The goal of our endeavor was to find out whether short peptides could be used as a dissolving factor for such crystals. In order to identify peptides able to dissolve crystals of calcium pyrophosphate, we screened through a random library of peptides using a phage display. The first screening was designed to select phages able to bind the acidic part of alendronic acid (pyrophosphate analog). The second was a catalytic assay in the presence of crystals. The best-performing peptides were subsequently chemically synthesized and rechecked for catalytic properties. One peptide, named R25, turned out to possess some hydrolytic activity toward crystals. Its catalysis is Mg2+-dependent and also works against soluble species of pyrophosphate.

Polyscias filicifolia (Araliaceae) Hairy Roots with Antigenotoxic and Anti-Photogenotoxic Activity.

Hairy root cultures are considered as a valuable source of bioactive phytoconstituents with expanding applicability for their production. In the present study, hairy root cultures of Polyscias filicifolia (Araliaceae), a traditional Southeast Asian medicinal plant, were established. The transformation with Agrobacterium rhizogenes ATCC 15834 allowed to obtain 15 root lines. The K-1 line, demonstrating the highest growth capabilities, was subjected to further investigations. To enhance the biosynthetic potential of hairy roots, methyl jasmonate elicitation approach was applied (MeJA; at different doses and exposure time), with subsequent transfer of elicited roots to control medium. This strategy resulted in chlorogenic acid production up to 1.59 mg/g dry weight. HPLC-PDA-ESI-MS analysis demonstrated variation in extracts composition and allowed to identify different caffeic and ferulic acid derivatives. Next, cytotoxic, antigenotoxic, and anti-photogenotoxic properties of hairy roots extracts were determined. None of the tested extracts were cytotoxic. In addition, they demonstrated significant antigenotoxic activity with the highest protective potential; up to 52% and 49% of inhibition of induction ratio (IR) induced by the 2-aminoanthracene was revealed for extracts derived from hairy roots elicited for 3 days with 50 µM MeJA and roots elicited for 7 days with 100 µM MeJA and then transferred for 30 days to control medium, respectively. These same extracts exhibited the highest anti-photogenotoxic potential, up to 36% of inhibition of chloropromazine-induced genotoxicity.

The role of drugs and selected dietary factors in cutaneous squamous cell carcinogenesis.

Cutaneous squamous cell carcinoma represents the second most common non-melanoma skin cancer and its incidence increases worldwide. This review provides an overview of selected exogenous risk factors for cutaneous squamous cell carcinoma, which include drugs (azathioprine, calcineurin inhibitors, hydrochlorothiazide, angiotensin-converting-enzyme inhibitors) and few dietary factors (fat meet, whole milk products, arsenic) to better understand squamous skin cancer etiopathogenesis. Ingredients such as leafy vegetables, nuts, fish, caffeine, niacin are preventive factors for cutaneous squamous cell cancer. The heart transplant recipients have an increased risk of squamous cell carcinoma development than kidney or liver transplant ones and switching photosensitizing azathioprine to mycophenolate mofetil can reduce the incidence of cutaneous squamous cell carcinoma. The great attention should be paid to early change of cardiac photosensitizing antihypertensive drugs to non-photosensitizing ones among patients with a history of prior skin cancers and among organ transplant recipients. Based on current knowledge that ultra-violet radiation is the main risk factor for squamous cell carcinoma development, promotion of the skin self-examination, photoprotection, tanning bed avoidance and early skin cancer diagnosis is important for this tumour prevention.

In Vivo, In Vitro and In Silico Studies of the Hybrid Compound AA3266, an Opioid Agonist/NK1R Antagonist with Selective Cytotoxicity.

AA3266 is a hybrid compound consisting of opioid receptor agonist and neurokinin-1 receptor (NK1R) antagonist pharmacophores. It was designed with the desire to have an analgesic molecule with improved properties and auxiliary anticancer activity. Previously, the compound was found to exhibit high affinity for µ- and δ-opioid receptors, while moderate binding to NK1R. In the presented contribution, we report on a deeper investigation of this hybrid. In vivo, we have established that AA3266 has potent antinociceptive activity in acute pain model, comparable to that of morphine. Desirably, with prolonged administration, our hybrid induces less tolerance than morphine does. AA3266, contrary to morphine, does not cause development of constipation, which is one of the main undesirable effects of opioid use. In vitro, we have confirmed relatively strong cytotoxic activity on a few selected cancer cell lines, similar to or greater than that of a reference NK1R antagonist, aprepitant. Importantly, our compound affects normal cells to smaller extent what makes our compound more selective against cancer cells. In silico methods, including molecular docking, molecular dynamics simulations and fragment molecular orbital calculations, have been used to investigate the interactions of AA3266 with MOR and NK1R. Insights from these will guide structural optimization of opioid/antitachykinin hybrid compounds.

Urea-Peptide Hybrids as VEGF-A165/NRP-1 Complex Inhibitors with Improved Receptor Affinity and Biological Properties.

Neuropilin-1 (NRP-1), the major co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2), may also independently act with VEGF-A165 to stimulate tumour growth and metastasis. Therefore, there is great interest in compounds that can block VEGF-A165/NRP-1 interaction. Peptidomimetic type inhibitors represent a promising strategy in the treatment of NRP-1-related disorders. Here, we present the synthesis, affinity, enzymatic stability, molecular modeling and in vitro binding evaluation of the branched urea-peptide hybrids, based on our previously reported Lys(hArg)-Dab-Oic-Arg active sequence, where the Lys(hArg) branching has been modified by introducing urea units to replace the peptide bond at various positions. One of the resulting hybrids increased the affinity of the compound for NRP-1 more than 10-fold, while simultaneously improving resistance for proteolytic stability in serum. In addition, ligand binding to NRP-1 induced rapid protein stock exocytotic trafficking to the plasma membrane in breast cancer cells. Examined properties characterize this compound as a good candidate for further development of VEGF165/NRP-1 inhibitors.

Huperzine A and Huperzine B Production by Prothallus Cultures of Huperzia selago (L.) Bernh. ex Schrank et Mart.

This is the first report of an efficient and effective procedure to optimize the biosynthesis of huperzine A (HupA) and huperzine B (HupB) in vitro from Huperzia selago gametophytes. Axenic tissue cultures were established using spores collected from the sporophytes growing in the wild. The prothalia were obtained after 7-18 months. Approximately 90 up to 100% of the gametophytes were viable and grew rapidly after each transfer on to a fresh medium every 3 months. The best biomass growth index for prothallus calculated on a fresh (FW) and dry weight (DW) basis, at 24 weeks of culture, was 2500% (FW) and 2200% (DW), respectively. The huperzine A content in the gametophytes was very high and ranged from 0.74 mg/g to 4.73 mg/g DW. The highest yield HupA biosynthesis at >4 mg/g DW was observed on W/S medium without growth regulators at 8 to 24 weeks of culture. The highest HupB content ranged from 0.10 mg/g to 0.52 mg/g DW and was obtained on the same medium. The results demonstrate the superiority of H. selago gametophyte cultures, with the level of HupA biosynthesis approximately 42% higher compared to sporophyte cultures and 35-fold higher than when the alkaloid was isolated from H. serrata, its current source for the pharmaceutical industry. Moreover, the biosynthesis of HupB was several-fold more efficient than in H. selago sporophytes growing in the wild. HPLC-HR-MS analyses of the extracts identified eight new alkaloids previously unreported in H. selago: deacetylfawcettine, fawcettimine, 16-hydroxyhuperzine B, deacetyllycoclavine, annopodine, lycopecurine, des-N-methylfastigiatine and flabelline.

ß2-Homo-Amino Acid Scan of µ-Selective Opioid Tetrapeptide TAPP.

TAPP (H-Tyr-d-Ala-Phe-Phe-NH2) is a potent, µ-selective opioid ligand. In order to gain further insights into pharmacophoric features of this tetrapeptide, we have performed a ß2-Homo-amino acid (ß2hAA) scan of the TAPP sequence. To this aim, 10 novel analogues have been synthesized and evaluated for µ-opioid and δ-opioid receptor affinity as well as for stability in human plasma. The derivatives included compounds in which a (R)- or (S)-ß2-Homo-Homologue replaced the amino acids in the TAPP sequence. The derivatives with (R)- or (S)-ß2hPhe4 turned out to bind µOR with affinities equal to that of the parent. ß2hAAs in position 1 and 3 resulted in rather large affinity decreases, but the change differed depending on the stereochemistry. ß2-Homologation in the second position gave derivatives with very poor µOR binding. According to molecular modelling, the presented α/ß-peptides adopt a variety of binding poses with their common element being an ionic interaction between a protonable amine of the first residue and Asp147. A feature required for high µOR affinity seems the ability to accommodate the ring in the fourth residue in a manner similar to that found for TAPP. Contrary to what might be expected, several compounds were significantly less stable in human plasma than the parent compound.

Antinociceptive and Cytotoxic Activity of Opioid Peptides with Hydrazone and Hydrazide Moieties at the C-Terminus.

In the present contribution, we analyze the influence that C-terminal extension of short opioid peptide sequences by organic fragments has on receptor affinity, in vivo analgesic activity, and antimelanoma properties. The considered fragments were based on either N-acylhydrazone (NAH) or N'-acylhydrazide motifs combined with the 3,5-bis(trifluoromethyl)phenyl moiety. Eleven novel compounds were synthesized and subject to biological evaluation. The analyzed compounds exhibit a diversified range of affinities for the µ opioid receptor (MOR), rather low δ opioid receptor (DOR) affinities, and no appreciable neurokinin-1 receptor binding. In three out of four pairs, N-acylhydrazone-based derivatives bind MOR better than their N'-acylhydrazide counterparts. The best of the novel derivatives have similar low nanomolar MOR binding affinity as the reference opioids, such as morphine and biphalin. The obtained order of MOR affinities was compared to the results of molecular docking. In vivo, four tested compounds turned out to be relatively strong analgesics. Finally, the NAH-based analogues reduce the number of melanoma cells in cell culture, while their N'-acylhydrazide counterparts do not. The antimelanoma properties are roughly correlated to the lipophilicity of the compounds.

Radiochemical Synthesis and Evaluation of Novel Radioconjugates of Neurokinin 1 Receptor Antagonist Aprepitant Dedicated for NK1R-Positive Tumors.

Aprepitant, a lipophilic and small molecular representative of neurokinin 1 receptor antagonists, is known for its anti-proliferative activity on numerous cancer cell lines that are sensitive to Substance P mitogen action. In the presented research, we developed two novel structural modifications of aprepitant to create aprepitant conjugates with different radionuclide chelators. All of them were radiolabeled with 68Ga and 177Lu radionuclides and evaluated in terms of their lipophilicity and stability in human serum. Furthermore, fully stable conjugates were examined in molecular modelling with a human neurokinin 1 receptor structure and in a competitive radioligand binding assay using rat brain homogenates in comparison to the aprepitant molecule. This initial research is in the conceptual stage to give potential theranostic-like radiopharmaceutical pairs for the imaging and therapy of neurokinin 1 receptor-overexpressing cancers.

Urea moiety as amide bond mimetic in peptide-like inhibitors of VEGF-A165/NRP-1 complex.

NRP-1 is an important co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2). Many reports suggested that NRP-1 might also serve as a separate receptor for VEGF-A165 causing stimulation of tumour growth and metastasis. Therefore, compounds interfering with VEGF-A165/NRP-1 complex triggered interest in the design of new molecules, including peptides, as anti-angiogenic and anti-tumour drugs. Here, we report the synthesis, affinity and stability evaluation of the urea-peptide hybrids, based on general Lys(hArg)-AA2-AA3-Arg sequence, where hArg residue was substituted by Arg urea unit. Such substitution does not substantially affected affinity of compounds for NRP-1 but significantly increased their proteolytic stability in plasma.

Fentanyl Family at the Mu-Opioid Receptor: Uniform Assessment of Binding and Computational Analysis.

Interactions of 21 fentanyl derivatives with µ-opioid receptor (µOR) were studied using experimental and theoretical methods. Their binding to µOR was assessed with radioligand competitive binding assay. A uniform set of binding affinity data contains values for two novel and one previously uncharacterized derivative. The data confirms trends known so far and thanks to their uniformity, they facilitate further comparisons. In order to provide structural hypotheses explaining the experimental affinities, the complexes of the studied derivatives with µOR were modeled and subject to molecular dynamics simulations. Five common General Features (GFs) of fentanyls' binding modes stemmed from these simulations. They include: GF1) the ionic interaction between D147 and the ligands' piperidine NH⁺ moiety; GF2) the N-chain orientation towards the µOR interior; GF3) the other pole of ligands is directed towards the receptor outlet; GF4) the aromatic anilide ring penetrates the subpocket formed by TM3, TM4, ECL1 and ECL2; GF5) the 4-axial substituent (if present) is directed towards W318. Except for the ionic interaction with D147, the majority of fentanyl-µOR contacts is hydrophobic. Interestingly, it was possible to find nonlinear relationships between the binding affinity and the volume of the N-chain and/or anilide's aromatic ring. This kind of relationships is consistent with the apolar character of interactions involved in ligand⁻receptor binding. The affinity reaches the optimum for medium size while it decreases for both large and small substituents. Additionally, a linear correlation between the volumes and the average dihedral angles of W293 and W133 was revealed by the molecular dynamics study. This seems particularly important, as the W293 residue is involved in the activation processes. Further, the Y326 (OH) and D147 (Cγ) distance found in the simulations also depends on the ligands' size. In contrast, neither RMSF measures nor D114/Y336 hydrations show significant structure-based correlations. They also do not differentiate studied fentanyl derivatives. Eventually, none of 14 popular scoring functions yielded a significant correlation between the predicted and observed affinity data (R < 0.30, n = 28).

Triazolopeptides Inhibiting the Interaction between Neuropilin-1 and Vascular Endothelial Growth Factor-165.

Inhibiting the interaction of neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has become an interesting mechanism for potential anticancer therapies. In our previous works, we have obtained several submicromolar inhibitors of this interaction, including branched pentapeptides of general structure Lys(Har)-Xxx-Xxx-Arg. With the intent to improve the proteolytic stability of our inhibitors, we turned our attention to 1,4-disubstituted 1,2,3-triazoles as peptide bond isosteres. In the present contribution, we report the synthesis of 23 novel triazolopeptides along with their inhibitory activity. The compounds were synthesized using typical peptide chemistry methods, but with a conversion of amine into azide completely on solid support. The inhibitory activity of the synthesized derivatives spans from 9.2% to 58.1% at 10 µM concentration (the best compound Lys(Har)-GlyΨ[Trl]GlyΨ[Trl]Arg, 3, IC50 = 8.39 µM). Synthesized peptidotriazoles were tested for stability in human plasma and showed remarkable resistance toward proteolysis, with half-life times far exceeding 48 h. In vitro cell survival test resulted in no significant impact on bone marrow derived murine cells 32D viability. By means of molecular dynamics, we were able to propose a binding mode for compound 3 and discuss the observed structure-activity relationships.

Ischemia/Reperfusion-Induced Translocation of PKCßII to Mitochondria as an Important Mediator of a Protective Signaling Mechanism in an Ischemia-Resistant Region of the Hippocampus.

Emerging reports indicate that activated PKC isoforms that translocate to the mitochondria are pro- or anti-apoptotic to mitochondrial function. Here, we concentrate on the role of PKCß translocated to mitochondria in relation to the fate of neurons following cerebral ischemia. As we have demonstrated previously ischemia/reperfusion injury (I/R) results in translocation of PKCß from cytoplasm to mitochondria, but only in ischemia-resistant regions of the hippocampus (CA2-4, DG), we hypothesize that this translocation may be a mediator of a protective signaling mechanism in this region. We have therefore sought to demonstrate a possible relationship between PKCßII translocation and ischemic resistance of CA2-4, DG. Here, we reveal that I/R injury induces a marked elevation of PKCßII protein levels, and consequent enzymatic activity, in CA2-4, DG in the mitochondrial fraction. Moreover, the administration of an isozyme-selective PKCßII inhibitor showed inhibition of I/R-induced translocation of PKCßII to the mitochondria and an increase in neuronal death following I/R injury in CA1 and CA2-4, DG in both an in vivo and an in vitro model of ischemia. The present results suggest that PKCßII translocated to mitochondria is involved in providing ischemic resistance of CA2-4, DG. However, the exact mechanisms by which PKCßII-mediated neuroprotection is achieved are in need of further elucidation.