Therapeutic Potential of Targeting Prokineticin Receptors in Diseases.

The prokineticins (PKs) were discovered approximately 20 years ago as small peptides inducing gut contractility. Today, they are established as angiogenic, anorectic, and proinflammatory cytokines, chemokines, hormones, and neuropeptides involved in variety of physiologic and pathophysiological pathways. Their altered expression or mutations implicated in several diseases make them a potential biomarker. Their G-protein coupled receptors, PKR1 and PKR2, have divergent roles that can be therapeutic target for treatment of cardiovascular, metabolic, and neural diseases as well as pain and cancer. This article reviews and summarizes our current knowledge of PK family functions from development of heart and brain to regulation of homeostasis in health and diseases. Finally, the review summarizes the established roles of the endogenous peptides, synthetic peptides and the selective ligands of PKR1 and PKR2, and nonpeptide orthostatic and allosteric modulator of the receptors in preclinical disease models. The present review emphasizes the ambiguous aspects and gaps in our knowledge of functions of PKR ligands and elucidates future perspectives for PK research. SIGNIFICANCE STATEMENT: This review provides an in-depth view of the prokineticin family and PK receptors that can be active without their endogenous ligand and exhibits "constitutive" activity in diseases. Their non- peptide ligands display promising effects in several preclinical disease models. PKs can be the diagnostic biomarker of several diseases. A thorough understanding of the role of prokineticin family and their receptor types in health and diseases is critical to develop novel therapeutic strategies with safety concerns.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL.

Dysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5' untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

eIF4A1 Is a Prognostic Marker and Actionable Target in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a primary liver tumor with high lethality and increasing incidence worldwide. While tumor resection or liver transplantation is effective in the early stages of the disease, the therapeutic options for advanced HCC remain limited and the benefits are temporary. Thus, novel therapeutic targets and more efficacious treatments against this deadly cancer are urgently needed. Here, we investigated the pathogenetic and therapeutic role of eukaryotic initiation factor 4A1 (eIF4A1) in this tumor type. We observed consistent eIF4A1 upregulation in HCC lesions compared with non-tumorous surrounding liver tissues. In addition, eIF4A1 levels were negatively correlated with the prognosis of HCC patients. In HCC lines, the exposure to various eIF4A inhibitors triggered a remarkable decline in proliferation and augmented apoptosis, paralleled by the inhibition of several oncogenic pathways. Significantly, anti-growth effects were achieved at nanomolar concentrations of the eIF4A1 inhibitors and were further increased by the simultaneous administration of the pan mTOR inhibitor, Rapalink-1. In conclusion, our results highlight the pathogenetic relevance of eIF4A1 in HCC and recommend further evaluation of the potential usefulness of pharmacological combinations based on eIF4A and mTOR inhibitors in treating this aggressive tumor.

Fluorizoline Blocks the Interaction between Prohibitin-2 and γ-Glutamylcyclotransferase and Induces p21Waf1/Cip1 Expression in MCF7 Breast Cancer Cells.

Prohibitin-2 (PHB2) is a scaffold protein that has pleiotropic functions, which include interacting with γ-glutamylcyclotransferase (GGCT) in the cytoplasm and repressing the transcriptional activities of the p21Waf1/Cip (p21) gene in the nucleus. The cytotoxic drug fluorizoline binds to PHB1/2 and exerts antiproliferative actions on cancer cells. However, the precise mechanism underlying the antiproliferative effects of fluorizoline is not fully elucidated. In the present study, we first show that fluorizoline induces p21 expression in several human cancer cell lines, including MCF7 breast cancer cells. Treatment of MCF7 cells with fluorizoline suppressed proliferation and prevented cells from entering into the DNA synthesis phase. Knockdown of p21 rescued the suppressed proliferation, indicating that fluorizoline inhibited MCF7 cell growth via the induction of p21. Overexpression of PHB2 in MCF7 cells prevented the induction of p21 expression by fluorizoline and restored the antiproliferative effects and blockade of cell cycle progression. Moreover, treatment of MCF7 cells with fluorizoline inhibited the interaction between endogenous PHB2 and GGCT proteins and reduced the level of nuclear localization of PHB2 proteins. These results indicate that targeting PHB2 with fluorizoline induces the expression of p21 and consequently blocks proliferation of cancer cells. SIGNIFICANCE STATEMENT: This study shows that fluorizoline may be a promising novel anticancer drug candidate that induces p21 expression and blocks cell-cycle progression in human cancer cell lines. In addition, we show that fluorizoline inhibits the interaction between PHB2 and GGCT and reduces the nuclear localization of PHB2 proteins.

A subset of flavaglines inhibits KRAS nanoclustering and activation.

The RAS oncogenes are frequently mutated in human cancers and among the three isoforms (KRAS, HRAS and NRAS), KRAS is the most frequently mutated oncogene. Here, we demonstrate that a subset of flavaglines, a class of natural anti-tumour drugs and chemical ligands of prohibitins, inhibit RAS GTP loading and oncogene activation in cells at nanomolar concentrations. Treatment with rocaglamide, the first discovered flavagline, inhibited the nanoclustering of KRAS, but not HRAS and NRAS, at specific phospholipid-enriched plasma membrane domains. We further demonstrate that plasma membrane-associated prohibitins directly interact with KRAS, phosphatidylserine and phosphatidic acid, and these interactions are disrupted by rocaglamide but not by the structurally related flavagline FL1. Depletion of prohibitin-1 phenocopied the rocaglamide-mediated effects on KRAS activation and stability. We also demonstrate that flavaglines inhibit the oncogenic growth of KRAS-mutated cells and that treatment with rocaglamide reduces non-small-cell lung carcinoma (NSCLC) tumour nodules in autochthonous KRAS-driven mouse models without severe side effects. Our data suggest that it will be promising to further develop flavagline derivatives as specific KRAS inhibitors for clinical applications.

The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis.

Fluorizoline (FLZ) binds to prohibitin-1 and -2 (PHB1/2), which are pleiotropic scaffold proteins known to affect signaling pathways involved in several intracellular processes. However, it is not yet clear how FLZ exerts its effect. Here, we show that exposure of three different human cancer cell lines to FLZ increases the phosphorylation of key translation factors, particularly of initiation factor 2 (eIF2) and elongation factor 2 (eEF2), modifications that inhibit their activities. FLZ also impaired signaling through mTOR complex 1, which also regulates the translational machinery, e.g. through the eIF4E-binding protein 4E-BP1. In line with these findings, FLZ potently inhibited protein synthesis. We noted that the first phase of this inhibition involves very rapid eEF2 phosphorylation, which is catalyzed by a dedicated Ca2+-dependent protein kinase, eEF2 kinase (eEF2K). We also demonstrate that FLZ induces a swift and marked rise in intracellular Ca2+ levels, likely explaining the effects on eEF2. Disruption of normal Ca2+ homeostasis can also induce endoplasmic reticulum stress, and our results suggest that induction of this stress response contributes to the increased phosphorylation of eIF2, likely because of activation of the eIF2-modifying kinase PKR-like endoplasmic reticulum kinase (PERK). We show that FLZ induces cancer cell death and that this effect involves contributions from the phosphorylation of both eEF2 and eIF2. Our findings provide important new insights into the biological effects of FLZ and thus the roles of PHBs, specifically in regulating Ca2+ levels, cellular protein synthesis, and cell survival.

Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases.

Over the last three decades, the scaffold proteins prohibitins-1 and -2 (PHB1/2) have emerged as key signaling proteins regulating a myriad of signaling pathways in health and diseases. Small molecules targeting PHBs display promising effects against cancers, osteoporosis, inflammatory, cardiac and neurodegenerative diseases. This review provides an updated overview of the various classes of PHB ligands, with an emphasis on their mechanism of action and therapeutic potential. We also describe how these ligands have been used to explore PHB signaling in different physiological and pathological settings.

Prokineticin signaling in heart-brain developmental axis: Therapeutic options for heart and brain injuries.

Heart and brain development occur simultaneously during the embryogenesis, and both organ development and injuries are interconnected. Early neuronal and cardiac injuries share mutual cellular events, such as angiogenesis and plasticity that could either delay disease progression or, in the long run, result in detrimental health effects. For this reason, the common mechanisms provide a new and previously undervalued window of opportunity for intervention. Because angiogenesis, cardiogenesis and neurogenesis are essential for the development and regeneration of the heart and brain, we discuss therein the role of prokineticin as an angiogenic neuropeptide in heart-brain development and injuries. We focus on the role of prokineticin signaling and the effect of drugs targeting prokineticin receptors in neuroprotection and cardioprotection, with a special emphasis on heart failure, neurodegenerativParkinson's disease and ischemic heart and brain injuries. Indeed, prokineticin triggers common pro-survival signaling pathway in heart and brain. Our review aims at stimulating researchers and clinicians in neurocardiology to focus on the role of prokineticin signaling in the reciprocal interaction between heart and brain. We hope to facilitate the discovery of new treatment strategies, acting in both heart and brain degenerative diseases.

One-Pot Selective Saturation and Functionalization of Heteroaromatics Leading to Dihydropyridines and Dihydroquinolines.

A one-pot regioselective two C-C-bond-forming dearomatization of pyridines and quinolines is disclosed. Two 3,4-betaines are identified for the first time as very useful organic synthons in heterocyclic chemistry. Furthermore, the chemical reactivity of the prepared trifluoromethyl ketones, a new type of push-pull enones, has been explored to develop straightforward methods for their functionalization. This protocol represents a breakthrough in the dearomatization of heteroaromatics as both the selective saturation and functionalization of heteroaromatics are achieved in high efficiency by the attachment of two substituents, including the valuable trifluoromethyl ketone group.

SFPH proteins as therapeutic targets for a myriad of diseases.

The stomatin/prohibitin/flotillin/HflK/HflC (SPFH) domain is present in an evolutionarily conserved family of proteins that regulate a myriad of signaling pathways in archaea, bacteria and eukaryotes. The most studied SPFH proteins, prohibitins, have already been targeted by different families of small molecules to induce anticancer, cardioprotective, anti-inflammatory, antiviral, and antiosteoporotic activities. Ligands of other SPFH proteins have also been identified and shown to act as anesthetics, anti-allodynia, anticancer, and anti-inflammatory agents. These findings indicate that modulators of human or bacterial SPFH proteins can be developed to treat a wide variety of human disorders.

eIF4F is a nexus of resistance to anti-BRAF and anti-MEK cancer therapies.

In BRAF(V600)-mutant tumours, most mechanisms of resistance to drugs that target the BRAF and/or MEK kinases rely on reactivation of the RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) signal transduction pathway, on activation of the alternative, PI(3)K-AKT-mTOR, pathway (which is ERK independent) or on modulation of the caspase-dependent apoptotic cascade. All three pathways converge to regulate the formation of the eIF4F eukaryotic translation initiation complex, which binds to the 7-methylguanylate cap (m(7)G) at the 5' end of messenger RNA, thereby modulating the translation of specific mRNAs. Here we show that the persistent formation of the eIF4F complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, is associated with resistance to anti-BRAF, anti-MEK and anti-BRAF plus anti-MEK drug combinations in BRAF(V600)-mutant melanoma, colon and thyroid cancer cell lines. Resistance to treatment and maintenance of eIF4F complex formation is associated with one of three mechanisms: reactivation of MAPK signalling, persistent ERK-independent phosphorylation of the inhibitory eIF4E-binding protein 4EBP1 or increased pro-apoptotic BCL-2-modifying factor (BMF)-dependent degradation of eIF4G. The development of an in situ method to detect the eIF4E-eIF4G interactions shows that eIF4F complex formation is decreased in tumours that respond to anti-BRAF therapy and increased in resistant metastases compared to tumours before treatment. Strikingly, inhibiting the eIF4F complex, either by blocking the eIF4E-eIF4G interaction or by targeting eIF4A, synergizes with inhibiting BRAF(V600) to kill the cancer cells. eIF4F not only appears to be an indicator of both innate and acquired resistance but also is a promising therapeutic target. Combinations of drugs targeting BRAF (and/or MEK) and eIF4F may overcome most of the resistance mechanisms arising in BRAF(V600)-mutant cancers.

Prokineticin-2 promotes chemotaxis and alternative A2 reactivity of astrocytes.

Astrocyte reactivity is disease- and stimulus-dependent, adopting either a proinflammatory A1 phenotype or a protective, anti-inflammatory A2 phenotype. Recently, we demonstrated, using cell culture, animal models and human brain samples, that dopaminergic neurons produce and secrete higher levels of the chemokine-like signaling protein Prokineticin-2 (PK2) as a compensatory protective response against neurotoxic stress. As astrocytes express a high level of PK2 receptors, herein, we systematically characterize the role of PK2 in astrocyte structural and functional properties. PK2 treatment greatly induced astrocyte migration, which was accompanied by a shift in mitochondrial energy metabolism, a reduction in proinflammatory factors, and an increase in the antioxidant genes Arginase-1 and Nrf2. Overexpression of PK2 in primary astrocytes or in the in vivo mouse brain induced the A2 astrocytic phenotype with upregulation of key protective genes and A2 reactivity markers including Arginase-1 and Nrf2, PTX3, SPHK1, and TM4SF1. A small-molecule PK2 agonist, IS20, not only mimicked the protective effect of PK2 in primary cultures, but also increased glutamate uptake by upregulating GLAST. Notably, IS20 blocked not only MPTP-induced reductions in the A2 phenotypic markers SPHK1 and SCL10a6 but also elevation of the of A1 marker GBP2. Collectively, our results reveal that PK2 regulates a novel neuron-astrocyte signaling mechanism by promoting an alternative A2 protective phenotype in astrocytes, which could be exploited for development of novel therapeutic strategies for PD and other related chronic neurodegenerative diseases. PK2 signals through its receptors on astrocytes and promotes directed chemotaxis. PK2-induced astrocyte reactivity leads to an increase in antioxidant and anti-inflammatory proteins while increasing glutamate uptake, along with decreased inflammatory factors. © 2018 Wiley Periodicals, Inc.

Scalable 9-Step Synthesis of the Splicing Modulator NVS-SM2.

NVS-SM2, the first activator of pre-mRNA splicing, displays remarkable pharmacological in vivo activities in models of spinal muscular atrophy. Herein we describe an improved approach to the synthesis of this compound, which features a convenient introduction of sterically encumbered amine moiety onto a fluoropyridazine intermediate.

Pro-differentiating effects of a synthetic flavagline on human teratocarcinomal cancer stem-like cells.

As initiators of the carcinogenic process, cancer stem cells (CSCs) are considered as new targets for anti-cancer therapies. However, these cells are hidden in the cancer bulk and remain relatively insensitive to chemotherapy, which targets their proliferative capacities. Alternatively, growing evidences have pointed out that a differentiation therapy could adversely affect these cells, which consequently should lose their self-renewal properties and become less aggressive. In order to evaluate the differentiation potential of an emerging class of anti-cancer drugs, we used the poorly differentiated teratocarcinomal cell as a model of Oct4-expressing CSC and determined the molecular mechanisms induced by the highly active flavagline FL3. The drug, administrated at sublethal concentration and for long period, was able to downregulate the expression levels of the stemness factors Oct4 and Nanog at both transcriptional and translational levels, concomitantly with a decrease of clonogenicity. The appearance of specific neural markers further demonstrated the differentiation properties of FL3. Interestingly, an expression of active caspase-3 and an upregulation of the expression of the germ cell nuclear factor were observed in treated cells; this suggests that the suppression of Oct4 expression required for the induction of differentiation involves overlapping mechanisms of protein degradation and gene repression. Finally, this study shows that FL3, like all-trans retinoic acid (ATRA), acts as a differentiation inducer of teratocarcinomal cells. Thus, FL3 offers an alternative possibility for cancer treatment since it could target the carcinogenic process by inducing the differentiation of ATRA-resistant and Oct4-expressing CSCs, without toxic side effects on normal cells.

Assessment of flavaglines as potential chikungunya virus entry inhibitors.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that recently caused large epidemics in islands in, and countries around, the Indian Ocean. There is currently no specific drug for therapeutic treatment or for use as a prophylactic agent against infection and no commercially available vaccine. Prohibitin has been identified as a receptor protein used by chikungunya virus to enter mammalian cells. Recently, synthetic sulfonyl amidines and flavaglines (FLs), a class of naturally occurring plant compounds with potent anti-cancer and cytoprotective and neuroprotective activities, have been shown to interact directly with prohibitin. This study therefore sought to determine whether three prohibitin ligands (sulfonyl amidine 1 m and the flavaglines FL3 and FL23) were able to inhibit CHIKV infection of mammalian Hek293T/17 cells. All three compounds inhibited infection and reduced virus production when cells were treated before infection but not when added after infection. Pretreatment of cells for only 15 minutes prior to infection followed by washing out of the compound resulted in significant inhibition of entry and virus production. These results suggest that further investigation of prohibitin ligands as potential Chikungunya virus entry inhibitors is warranted.

Novel carbocationic rearrangements of 1-styrylpropargyl alcohols.

The dehydration and subsequent cyclization reactions of 1-styrylpropargyl alcohols was examined. In the course of these studies, numerous scaffolds were synthesized, including a furan, a cyclopentenone, an acyclic enone and even a naphthalenone. The diversity of these structural motifs lies in novel cascades of reactions originating from a common carbocationic manifold.

Search for the most 'primitive' membranes and their reinforcers: a review of the polyprenyl phosphates theory.

Terpenoids have an essential function in present-day cellular membranes, either as membrane reinforcers in Eucarya and Bacteria or as principal membrane constituents in Archaea. We have shown that some terpenoids, such as cholesterol and α, ω-dipolar carotenoids reinforce lipid membranes by measuring the water permeability of unilamellar vesicles. It was possible to arrange the known membrane terpenoids in a 'phylogenetic' sequence, and a retrograde analysis led us to conceive that single-chain polyprenyl phosphates might have been 'primitive' membrane constituents. By using an optical microscopy, we have observed that polyprenyl phosphates containing 15 to 30 C-atoms form giant vesicles in water in a wide pH range. The addition of 10 % molar of some polyprenols to polyprenyl phosphate vesicles have been shown to reduce the water permeability of membranes even more efficiently than the equimolecular addition of cholesterol. A 'prebiotic' synthesis of C10 and C15 prenols from C5 monoprenols was achieved in the presence of a montmorillonite clay. Hypothetical pathway from C1 or C2 units to 'primitive' membranes and that from 'primitive' membranes to archaeal lipids are presented.

Loss of prohibitin 2 in Schwann cells dysregulates key transcription factors controlling developmental myelination.

Schwann cells are critical for the proper development and function of the peripheral nervous system, where they form a mutually beneficial relationship with axons. Past studies have highlighted that a pair of proteins called the prohibitins play major roles in Schwann cell biology. Prohibitins are ubiquitously expressed and versatile proteins. We have previously shown that while prohibitins play a crucial role in Schwann cell mitochondria for long-term myelin maintenance and axon health, they may also be present at the Schwann cell-axon interface during development. Here, we expand on this work, showing that drug-mediated modulation of prohibitins in vitro disrupts myelination and confirming that Schwann cell-specific ablation of prohibitin 2 (Phb2) in vivo results in early and severe defects in peripheral nerve development. Using a proteomic approach in vitro, we identify a pool of candidate PHB2 interactors that change their interaction with PHB2 depending on the presence of axonal signals. Furthermore, we show in vivo that loss of Phb2 in mouse Schwann cells causes ineffective proliferation and dysregulation of transcription factors EGR2 (KROX20), POU3F1 (OCT6) and POU3F2 (BRN2) that are necessary for proper Schwann cell maturation. Schwann cell-specific deletion of Jun, a transcription factor associated with negative regulation of myelination, confers partial rescue of the development defect seen in mice lacking Schwann cell Phb2. This work develops our understanding of Schwann cell biology, revealing that Phb2 may directly or indirectly modulate the timely expression of transcription factors necessary for proper peripheral nervous system development, and proposing candidates that may play a role in PHB2-mediated integration of axon signals in the Schwann cell.

Cytochromes P450 CYP94C1 and CYP94B3 catalyze two successive oxidation steps of plant hormone Jasmonoyl-isoleucine for catabolic turnover.

The jasmonate hormonal pathway regulates important defensive and developmental processes in plants. Jasmonoyl-isoleucine (JA-Ile) has been identified as a specific ligand binding the COI1-JAZ co-receptor to relieve repression of jasmonate responses. Two JA-Ile derivatives, 12OH-JA-Ile and 12COOH-JA-Ile, accumulate in wounded Arabidopsis leaves in a COI1- and JAR1-dependent manner and reflect catabolic turnover of the hormone. Here we report the biochemical and genetic characterization of two wound-inducible cytochromes P450, CYP94C1 and CYP94B3, that are involved in JA-Ile oxidation. Both enzymes expressed in yeast catalyze two successive oxidation steps of JA-Ile with distinct characteristics. CYP94B3 performed efficiently the initial hydroxylation of JA-Ile to 12OH-JA-Ile, with little conversion to 12COOH-JA-Ile, whereas CYP94C1 catalyzed preferentially carboxy-derivative formation. Metabolic analysis of loss- and gain-of-function plant lines were consistent with in vitro enzymatic properties. cyp94b3 mutants were largely impaired in 12OH-JA-Ile levels upon wounding and to a lesser extent in 12COOH-JA-Ile levels. In contrast, cyp94c1 plants showed wild-type 12OH-JA-Ile accumulation but lost about 60% 12COOH-JA-Ile. cyp94b3cyp94c1 double mutants hyperaccumulated JA-Ile with near abolition of 12COOH-JA-Ile. Distinct JA-Ile oxidation patterns in different plant genotypes were correlated with specific JA-responsive transcript profiles, indicating that JA-Ile oxidation status affects signaling. Interestingly, exaggerated JA-Ile levels were associated with JAZ repressor hyperinduction but did not enhance durably defense gene induction, revealing a novel negative feedback signaling loop. Finally, interfering with CYP94 gene expression affected root growth sensitivity to exogenous jasmonic acid. These results identify CYP94B3/C1-mediated oxidation as a major catabolic route for turning over the JA-Ile hormone.