Title: Pn-1, AN ANTINOCICEPTIVE PEPTIDE DERIVED FROM SPIDER TOXIN ACTING THROUGH CB1 CANNABINOID RECEPTORS

Author: Micheline F. Donato1, 2, Nayara M. Souza3, Jarbas R. Magalhães1, Moacyr Comar Jr.3, Wafa Hourani2, Stephen P. H. Alexander2, Maria Elena de Lima1,4, Published Year: 2018

GPCR Pharmacology - The Next Generation, University of Copenhagen, Denmark

Faculty:

Abstract: Cannabinoid receptors CB1 (CB1R) and CB2 (CB2R) are G-protein-coupled receptors (GPCRs). The stimulation of cannabinoid receptors in the brain may produce dysphoria, euphoria, hypothermia, appetite stimulation, memory impairment and analgesia [1]. The existence of an endogenous cannabinergic pain-modulatory system makes an attractive target in the search for new drugs, in particular when opiates are ineffective for pain treatment. Peptide toxins produced by a variety of organisms have evolved with different targets, including GPCR, enzymes and ion channels. Our group has previously reported synthetic peptides designed from the Brazilian wandering spider toxins. The synthetic nontoxic peptide PnPP-19 (BR 10 2012 020800-8 A2) induced antinociception involving cannabinoid and opioid systems [2]. The present study, antinociceptive synthetic peptide, Pn1 (11 amino acid residues, MW= 1.35 kDa) [3] was undertaken to investigate the interaction of Pn-1 with the CB1R and its pharmacological role on induced analgesia. Nociceptive thresholds were measured by the in vivo paw pressure test on male Wistar rats (180-200 g). Pn1 induced a dose-dependent response. Blocking opioid receptors failed to alter the antinociceptive activity. However, the blocked of CB1R at the highest dose of AM251 inhibited the antinociceptive effect. Also, inhibition of MAGL or the anandamide transporter potentiated the activity of the Pn-1. We evaluated in vitro the selectivity of Pn-1 using CHO-hCB1 and CHO-hCB2 receptors. At low concentrations, Pn-1 appeared to increase [35S]GTPγS binding and at concentrations of 10 μM and above, reduced it. In the presence of CP55940, it acted as an antagonist at high concentrations. Monitoring cAMP levels confirmed selective activity on CB1R. Pn-1 acts complexly at CB1 receptors.

Keywords: cannabinoids, opioid,Pn1,G-protein-coupled receptors (GPCRs)

Title: Coupling of the human recombinant CB2 cannabinoid receptor to extracellular signal-regulated kinase and calcium elevation in Chinese hamster ovary cells

Author: Wafa Hourani, Richard E Roberts and Stephen PH Alexander., Published Year: 2017

8th European Workshop on Cannabinoid Research, University of Roehampton, London, UK

Faculty:

Abstract: Background: CB1 cannabinoid receptors are characterised as coupling via the Gi/o family of G proteins to the inhibition of adenylyl cyclase activity and the activation of extracellular signal-regulated kinase (ERK)1. CB1 receptors in neurones also inhibit voltage-gated calcium channels, activate potassium channels and stimulate calcium mobilisation1. CB2 cannabinoid receptors are described to inhibit adenylyl cyclase and activate ERK, but not to regulate ion channel function1. In this study, we have investigated the coupling of human recombinant CB2 cannabinoid receptors expressed in Chinese hamster ovary cells. Methods: Human recombinant CB2 cannabinoid receptors were expressed in Chinese hamster ovary cells. ERK activation was assessed using in cell immunocytochemistry quantified with a Li-Cor Odyssey, while intracellular calcium levels were assessed using fluo-4 with a FlexStation with ATP as a positive control. Results:A range of cannabinoid agonists both endogenous (anandamide and 2-arachidonoylglycerol), natural (THC, β-caryophyllene and caryophyllene N-oxide) and synthetic (CP55940, WIN55,212-2, fenofibrate,HU210, JWH133, and JWH015) were tested. The following agents evoked maximal responses similar to the ATP (78-90 %) with the rank order: WIN55212-2 (pEC50 7.7 ± 0.1)> HU210 (7.4 ± 0.2)> fenofibrate (7.2 ± 0.1), JWH015 (7.2 ± 0.1)>2-AG (6.8 ± 0.1)>JWH133 (6.5 ± 0.1). AEA evoked a reduced maximal response (51%, 5.7 ± 0.2), while β-caryophyllene and caryophyllene N-oxide were inactive. 1 µM AM630, a CB2-selective antagonist, or pretreatment with 100 ng/mL pertussis toxin blocked responses to all these agonists. Calcium ion levels were stimulated by cannabinoid agonists, albeit in a slower manner than ATP. These responses were quantified as area under the curve: WIN55212-2 (21 ± 5 % ATP response, 7.3 ± 0.1) , HU210 (24 ± 3, 6.8 ± 0.3), CP55940 (37 ± 5, 6.7 ± 0.3), JWH015 (24 ± 3, 6.4 ± 0.1), fenofibrate (26 ± 3, 6.3 ± 0.2), 2-AG (34 ± 6, 6.2 ± 0.2), JWH133 (29 ± 7, 6.0 ± 0.1) , AEA (11 ± 1, 5.7 ± 0.3). Responses to THC failed to reach statistical significance, while those to β-caryophyllene and caryophyllene N-oxide were not distinguishable from background. Calcium responses to cannabinoid ligands were blocked by pretreatment with 1 µM AM630 or 100 ng/mL pertussis toxin. Cannabinoid ligand-evoked calcium responses were unaltered in the presence of 10 µM U0126, a selective inhibitor of ERK activation. Conclusions: This is the first study to determine coupling of the CB2 cannabinoid receptor to calcium elevation in recombinant expression.

Keywords: cannabinoid receptors,extracellular signal-regulated kinase,calcium

Title: COUPLING OF THE HUMAN RECOMBINANT CB2 CANNABINOID RECEPTOR TO AKT/PROTEIN KINASE B ACTIVATION/PHOSPHORYLATION IN CHINESE HAMSTER OVARY CELLS

Author: Wafa Hourani, Richard E Roberts and Stephen PH Alexander., Published Year: 2018

The 28th Annual International Cannabinoid Research Society Symposium on the Cannabinoids, Leiden university, the Netherlands

Faculty:

Abstract: Background: Cannabinoids affect the proliferation and differentiation of neural precursor cells in rats and mice. In oligodendrocytes, cannabinoid receptors influence stem cell survival and differentiation via the phosphatidylinositol 3-kinase:protein kinase B (PI3K-Akt, http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=285) signalling pathway(Gomez, Sanchez-Rodriguez et al. 2011). Recruitment to the plasma membrane and binding to phosphatidylinositol 3,4,5-trisphosphate allows phosphorylation and activation of Akt. The main objective of this investigation was to quantify the coupling of CB2 receptors to activation of the Akt signalling pathway. Methods: Chinese hamster ovary cells were stably transfected with human recombinant CB2 cannabinoid receptors. Activation of the Akt signalling pathway was assessed using a phosphopan-Akt antibody and in cell immunocytochemistry in 96-well microtitre plates quantified with a Licor Odyssey using fetal bovine serum as a positive control. Results: Multiple CB2 cannabinoid receptor agonists were investigated including the endogenous ligands; 2-AG and anandamide, as well as the synthetic ligands; CP55940, fenofibrate, HU210 ,WIN55,212-2, JWH015 and JWH133. A rank order of potency was CP55490 (pEC50 7.9 ± 0.04) > WIN55,212-2 (7.6 ± 0.1), HU210 (7.4 ± 0.1) > fenofibrate (7.1 ± 0.1), JWH015 (7.0 ± 0.1) > 2- AG (6.7 ± 0.2), JWH133 (6.3 ± 0.1) > anandamide (5.8 ± 0.1). The maximal effect induced by the different agonists was indicated as the Emax relative to the FBS response with 2-AG producing a maximum response at 149 ± 4 %, HU210 (142 ± 10), CP55940 (139 ± 12), fenofibrate (139 ± 11), WIN55,212-2 (116 ± 10), JWH015 (100 ± 10), JWH133 (98 ± 7), anandamide (71 ± 13). The phosphorylation/activation of the Akt signalling pathway was significantly attenuated by pretreatment with the CB2 selective antagonist AM630 (1 µM) or pertussis toxin (100 ng/mL). Conclusion This is the first study to quantify coupling of the CB2 receptor to Akt phosphorylation using cell immunocytochemistry. In common with other signalling pathways, 2-AG appears to act as a full agonist, while anandamide is a partial agonist, in the phosphorylation of Akt.

Keywords: Cannabinoids, cannabinoid receptors,anandamide, protein kinase B

Title: Cannabinoid ligands, receptors and enzymes: Pharmacological tools and therapeutic potential

Author: Wafa Hourani, Stephen P. H. Alexander, Published Year: 2018

Brain and Neuroscience Advances, Volume 2: 18

Faculty:

Abstract: Endocannabinoids have been identified to have roles in numerous physiological and pathological processes. Largely due to the association of the effects of Cannabis administration on mental states, the CNS impact of the endocannabinoid system has been the most intensively studied. Here, we provide a brief summary of the endocannabinoid system, comprising the receptors and the multiple endogenous lipid derivatives which activate them, as well as the enzymes which control the levels of these lipid derivatives. We identify pharmacological tools which may be used to interrogate the endocannabinoid system, as well as current and future options to exploit the system in the clinic.

Keywords: Cannabis, cannabinoids, the endocannabinoid system, anandamide, 2-arachidonoylglycerol

Title: Antitubercular, Cytotoxicity, and Computational Target Validation of Dihydroquinazolinone Derivatives

Author: by Katharigatta N. Venugopala 1,2,*ORCID,Nizar A. Al-Shar’i 3,Lina A. Dahabiyeh 4ORCID,Wafa Hourani 5,Pran Kishore Deb 5,*ORCID,Melendhran Pillay 6,Bashaer Abu-Irmaileh 7,Yasser Bustanji 7,8,Sandeep Chandrashekharappa 9ORCID,Christophe Tratrat 1ORCID, Published Year: 2022

ANTIBIOTICS,

Faculty:

Abstract: A series of 2,3-dihydroquinazolin-4(1H)-one derivatives (3a3m) was screened for in vitro whole-cell antitubercular activity against the tubercular strain H37Rv and multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains. Compounds 3l and 3m with di-substituted aryl moiety (halogens) attached to the 2-position of the scaffold showed a minimum inhibitory concentration (MIC) of 2 µg/mL against the MTB strain H37Rv. Compound 3k with an imidazole ring at the 2-position of the dihydroquinazolin-4(1H)-one also showed significant inhibitory action against both the susceptible strain H37Rv and MDR strains with MIC values of 4 and 16 µg/mL, respectively. The computational results revealed the mycobacterial pyridoxal-5′-phosphate (PLP)-dependent aminotransferase (BioA) enzyme as the potential target for the tested compounds. In vitro, ADMET calculations and cytotoxicity studies against the normal human dermal fibroblast cells indicated the safety and tolerability of the test compounds 3k3m. Thus, compounds 3k3m warrant further optimization to develop novel BioA inhibitors for the treatment of drug-sensitive H37Rv and drug-resistant MTB

Keywords: Keywords: dihydroquinazolin-4(1H)-ones; anti-TB activity; MTT assay; molecular docking studies; molecular dynamic simulations studies

Title: Anti-tubercular activity and molecular docking studies of indolizine derivatives targeting mycobacterial InhA enzyme

Author: Katharigatta N. VenugopalaORCID Icon,Sandeep Chandrashekharappa,Pran Kishore DebORCID Icon,Christophe Tratrat,Melendhran Pillay,Deepak Chopra,Nizar A. Al-Shar’iORCID Icon,Wafa Hourani,Lina A. DahabiyehORCID Icon,Pobitra Borah,Rahul D. Nagdeve,Susanta, Published Year: 2021

Journal of Enzyme Inhibition and Medicinal Chemistry , Volume 36, 2021

Faculty:

Abstract: A series of 1,2,3-trisubstituted indolizines (2a2f, 3a3d, and 4a4c) were screened for in vitro whole-cell anti-tubercular activity against the susceptible H37Rv and multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains. Compounds 2b2d, 3a3d, and 4a4c were active against the H37Rv-MTB strain with minimum inhibitory concentration (MIC) ranging from 4 to 32 µg/mL, whereas the indolizines 4a4c, with ethyl ester group at the 4-position of the benzoyl ring also exhibited anti-MDR-MTB activity (MIC = 1664 µg/mL). In silico docking study revealed the enoyl-acyl carrier protein reductase (InhA) and anthranilate phosphoribosyltransferase as potential molecular targets for the indolizines. The X-ray diffraction analysis of the compound 4b was also carried out. Further, a safety study (in silico and in vitro) demonstrated no toxicity for these compounds. Thus, the indolizines warrant further development and may represent a novel promising class of InhA inhibitors and multi-targeting agents to combat drug-sensitive and drug-resistant MTB strains.

Keywords: : Indolizinemycobacterium tuberculosisInhAdockingX-ray crystal structure

Title: A NOVEL BRAZILIAN SPIDER TOXIN ANALOGUE IS ANTINOCICEPTIVE ACTING VIA THE CANNABINOID SYSTEM

Author: Micheline F. Donato*,1, 2, Nayara M. Souza3 , Wafa Hourani2 , Moacyr Comar Jr.3 , Stephen P. H. Alexander2 , Jarbas R. Magalhães3 and Maria Elena de Lima, Published Year: 2018

The 28th Annual International Cannabinoid Research Society Symposium on the Cannabinoids, Leiden university, the Netherlands

Faculty:

Abstract: Peptide toxins produced by a variety of organisms have evolved with different targets, including GPCR, enzymes and ion channels, where there are many examples of ligands with high potency and selectivity. The Brazilian scientific team developed peptide analogues of an active component of the venom of the Brazilian wandering spider (Phoneutria nigriventer). The synthetic nontoxic peptide PnPP-19 (BR 10 2012 020800-8 A2) induced antinociception involving the inhibition of neutral endopeptidase and activation of CB1 cannabinoid receptors as well as µ and δ opioid receptors (Freitas, Br J Pharmacol PMID: 26947933). In this work, a novel synthetic nontoxic peptide, Pn1 (11 amino acid residues, MW= 1.35 kDa, in patent application) modeled from δCtenitoxin-Pn1a toxin was examined. Nociceptive thresholds were measured by the in vivo paw pressure test on male Wistar rats (180- 200 g). First, to investigate the role of Pn1 in nociception, the peptide was injected (1.5, 2.5, 5 and 10 µg per paw) or vehicle (50 µl) into rat paws-that were hyperalgesic following the administration of carrageenan (250 µg). Pn1 induced a dose-dependent antinociceptive response, with the maximal effects at 10 µg per paw, which peaked at 30 min post-administration. The role of the CB1 cannabinoid receptor in these effects was investigated with intraplantar co-administration of AM251 (40, 80 and 160 µg per paw). The highest dose totally inhibited the antinociceptive effect of Pn1 (10 µg per paw). Moreover, we investigated whether enzymatic degradation by FAAH, MAGL and anandamide endocannabinoid transporter pathways could be contributing to the antinociceptive activity using intraplantar administration of JZL184 (14 µg per paw); MAFP, (4 µg per paw); or VDM11 (40 µg per paw). The results showed that inhibition of MAGL or the transporter potentiated the antinociceptive activity of the Pn-1. In in vitro studies, we evaluated the selectivity of Pn1 using CHO-CB1 and CHO-CB2 human receptors and [35S]-GTPγS binding assays. The preliminary results indicated that Pn1 (10 µM) appears to exhibit selectivity and affinity for the CB1 receptor. Further studies of the downstream signalling pathways for the CB1 receptor (calcium elevation and ERK1/2 phosphorylation), however, failed to show significant effects of Pn1 (10 µM). Antinociception induced by Pn1 appears to involve the inhibition of MAGL and anandamide endocannabinoid transporter and the selective activation of CB1 receptors. This novel peptide could be useful as a new antinociceptive drug candidate