Cannabis sativa (Hemp) Seeds, Δ9-Tetrahydrocannabinol, and Potential Overdose.

Introduction:Cannabis sativa (hemp) seeds are popular for their high nutrient content, and strict regulations are in place to limit the amount of potentially harmful phytocannabinoids, especially Δ9-tetrahydrocannabinol (Δ9-THC). In Canada, this limit is 10 µg of Δ9-THC per gram of hemp seeds (10 ppm), and other jurisdictions in the world follow similar guidelines. Materials and Methods: We investigated three different brands of consumer-grade hemp seeds using four different procedures to extract phytocannabinoids, and quantified total Δ9-THC and cannabidiol (CBD). Discussion: We discovered that Δ9-THC concentrations in these hemp seeds could be as high as 1250% of the legal limit, and the amount of phytocannabinoids depended on the extraction procedure employed, Soxhlet extraction being the most efficient across all three brands of seeds. Δ9-THC and CBD exhibited significant variations in their estimated concentrations even from the same brand, reflecting the inhomogeneous nature of seeds and variability due to the extraction method, but almost in all cases, Δ9-THC concentrations were higher than the legal limit. These quantities of total Δ9-THC may reach as high as 3.8 mg per gram of hemp seeds, if one were consuming a 30-g daily recommended amount of hemp seeds, and is a cause for concern for potential toxicity. It is not clear if these high quantities of Δ9-THC are due to contamination of the seeds, or any other reason. Conclusion: Careful consideration of the extraction method is very important for the measurement of cannabinoids in hemp seeds.

Noncovalent Protein Arginine Deiminase (PAD) Inhibitors Are Efficacious in Animal Models of Multiple Sclerosis.

Peptidyl arginine deiminases have been shown to be hyperactive in neurodegenerative diseases including multiple sclerosis. An α-amino acid-based core structure, derived from a hydantoin core, with unique heterocycles on the side chains were synthesized as potential noncovalent inhibitors of PAD enzymes. Among the various heterocycles investigated, compound 23, carrying an imidazole moiety, exhibited the highest potency in this series with some selectivity for PAD2, and was further investigated in vivo. Pharmacokinetics in mice suggested the Cmax to be 12.0 ± 2.5 µg/mL and 170 ± 10 ng/mL in the serum and brain, respectively, when compound 23 was administered at 50 mg/kg via single dose ip. At the same dose, compound 23 also reversed physical disability and cleared the brain of T-cell infiltration in an EAE mouse model of multiple sclerosis (MS). This novel series of compounds show promise for further development as disease modifying agents for the potential treatment of MS.

A novel class of Plasmodial ClpP protease inhibitors as potential antimalarial agents.

The prokaryotic ATP-dependent ClpP protease, localized in the relict plastid of malaria parasite, represents a potential drug target. In the present study, we utilized in silico structure-based screening and medicinal chemistry approaches to identify a novel pyrimidine series of compounds inhibiting P. falciparum ClpP protease activity and evaluated their antiparasitic activities. Structure-activity relationship indicated that morpholine moiety at C2, an aromatic substitution at N3 and a 4-oxo moiety on the pyrimidine are important for potent inhibition of ClpP enzyme along with antiparasiticidal activity. Compound 33 exhibited potent antiparasitic activity (EC50 9.0±0.2µM), a 9-fold improvement over the antiparasitic activity of the hit molecule 6. Treatment of blood stage P. falciparum cultures with compound 33 caused morphological and developmental abnormalities in the parasites; further, compound 33 treatment hindered apicoplast development indicating the targeting of apicoplast.

Inhibitors of protein arginine deiminases and their efficacy in animal models of multiple sclerosis.

Protein arginine deiminases (PAD) are implicated in a variety of inflammatory and neurodegenerative diseases including multiple sclerosis (MS). Following the discovery of an in silico hit containing hydantoin and a piperidine moiety, we hypothesized that a 2-carbon linker on the hydantoin would be necessary for a 5-membered heterocycle for optimal PAD inhibitory activity. We designed thirteen compounds as potential inhibitors of PAD2 and PAD4 enzymes-two important PAD enzymes implicated in MS. Two compounds, one with an imidazole moiety (22) and the other with a tetrazole moiety (24) showed good inhibition of PAD isozymes in vitro and in the EAE mouse model of MS in vivo. Further experiments suggested that compound 22, a non-covalent inhibitor of PAD2 and PAD4, exhibits dose-dependent efficacy in the EAE mouse model and in the cuprizone-mediated demyelination model.

Small Molecule Agonists for the Type I Interferon Receptor: An In Silico Approach.

Type I interferons (IFNs) exhibit broad-spectrum antiviral activity, with potential utility against emerging acute virus infections that pose a threat to global health. Recombinant IFN-αs that have been approved for clinical use require cold storage and are administered through intramuscular or subcutaneous injection, features that are problematic for global distribution, storage, and administration. Cognizant that the biological potency of an IFN-α subtype is determined by its binding affinity to the type I IFN receptor, IFNAR, we identified a panel of small molecule nonpeptide compounds using an in silico screening strategy that incorporated specific structural features of amino acids in the receptor-binding domains of the most potent IFN-α, IFN alfacon-1. Hit compounds were selected based on ease of synthesis and formulation properties. In preliminary biological assays, we provide evidence that these compounds exhibit antiviral activity. This proof-of-concept study validates the strategy of in silico design and development for IFN mimetics.

Structure-activity relationships of pyrazole derivatives as potential therapeutics for immune thrombocytopenias.

Idiopathic or immune thrombocytopenia (ITP) is a serious clinical disorder involving the destruction of platelets by macrophages. Small molecule therapeutics are highly sought after to ease the burden on current therapies derived from human sources. Earlier, we discovered that dimers of five-membered heterocycles exhibited potential to inhibit phagocytosis of human RBCs by macrophages. Here, we reveal a structure-activity relationship of the bis-pyrazole class of molecules with -C-C-, -C-N- and -C-O- linkers, and their evaluation as inhibitors of phagocytosis of antibody-opsonized human RBCs as potential therapeutics for ITP. We have uncovered three potential candidates, 37, 47 and 50, all carrying a different linker connecting the two pyrazole moieties. Among these compounds, hydroxypyrazole derivative 50 is the most potent compound with an IC50 of 14 ± 9 µM for inhibiting the phagocytosis of antibody-opsonized human RBCs by macrophages. None of the compounds exhibited significant potential to induce apoptosis in peripheral blood mononuclear cells (PBMCs). Current study has revealed specific functional features, such as up to 2-atom spacer arm and alkyl substitution at one of the N(1) positions of the bivalent pyrazole core to be important for the inhibitory activity.

Small molecule mimetics of an interferon-α receptor interacting domain.

Small molecules that mimic IFN-α epitopes that interact with the cell surface receptor, IFNAR, would be useful therapeutics. One such 8-amino acid region in IFN-α2, designated IRRP-1, was used to derive 11 chemical compounds that belong to 5 distinct chemotypes, containing the molecular features represented by the key residues Leu30, Arg33, and Asp35 in IRRP-1. Three of these compounds exhibited potential mimicry to IRRP-1 and, in cell based assays, as predicted, effectively inhibited IFNAR activation by IFN-α. Of these, compound 3 did not display cell toxicity and reduced IFN-α-inducible STAT1 phosphorylation and STAT-DNA binding. Based on physicochemical properties' analyses, our data suggest that moieties with acidic pKa on the small molecule may be a necessary element for mimicking the carboxyl group of Asp35 in IRRP-1. Our data confirm the relevance of this strategy of molecular mimicry of ligand-receptor interaction domains of protein partners for small molecule drug discovery.

Antimalarial activities of 6-iodouridine and its prodrugs and potential for combination therapy.

Resistance by Plasmodium falciparum to almost all clinically used antimalarial drugs requires the development of new classes of antimalarials. 6-Iodouridine (15), a novel and potent inhibitor of orotidine 5'-monophosphate decarboxylase (ODCase), exhibited efficacy in a mouse model infected by P. chabaudi chabaudi. Compound 15 exhibited promising antimalarial activity against P. falciparum, including drug-resistant isolates, and no rapid drug-resistant populations of the parasite were observed when challenged with 15. Uridine provided options to overcome any toxicity in the host but still suppressing the parasite load when treated with 15. In drug combination studies, compound 15 showed good efficacy in vivo with artemisinin and azithromycin. The propionyl ester of 15 exhibited superior antimalarial efficacy. Antimalarial activities of 15 and its prodrugs and potential for combination therapy are discussed in the context of novel strategies.

Novel inhibitors of protein arginine deiminase with potential activity in multiple sclerosis animal model.

In multiple sclerosis (MS), myelin basic protein (MBP), critical for the maintenance of myelin compaction and protecting against degradation, is known to contain concentrations of the noncoded amino acid, "citrulline", in abnormal proportions. Peptidyl arginine deiminase (PAD) catalyzes the post-translational citrullination of proteins via the deimination of Arg residues. In the central nervous system, specifically PAD2 and PAD4, are the enzymes responsible for the citrullination. We used in silico screening of commercial libraries to find small molecules that would reversibly inhibit PAD4. An initial set of 10 diverse compounds was selected from the screen, and from these compounds, 3, 4, 6, and 8 showed promising inhibitory activities against PAD4 with Ki in the range of 115-153 µM. Compound 4 was selected to partake in an in vivo MOG EAE mouse model study to evaluate its effect in MS-like conditions. Results from the 24 day pilot mouse study showed an improved clinical outcome for mice being administered compound 4 compared to the control group. In brain, 4 treated mice showed a clear reduction in the CD3 +ve T cells. These results suggest that compound 4 may have potential utility and confirmed that noncovalent inhibitors of PAD enzymes can be developed as potential agents targeting MS pathology.

Interrogation of the Active Sites of Protein Arginine Deiminases (PAD1, -2, and -4) Using Designer Probes.

Protein arginine deiminases (PADs) are involved in a number of cellular pathways, and they catalyze the transformation of peptidyl arginine residue into a citrulline as part of post-translational modifications. To understand ligand preferences, a group of probe molecules were investigated against PAD1, PAD2, and PAD4. These probe molecules carried a well-known covalent modifier of the catalytic cysteine residue, 2-chloroacetamidine moiety, which was tethered to an α-amino acid via a carbon linker. The chain length for the linker varied from 0 to 4. Time-dependent assays indicated that 2-chloroacetamidine (2CA) with no linker inhibited all PAD enzymes with a similar trend in the second-order rate constants, although with poor affinity. Among the other three probe molecules, compound 3 with a three-carbon linker exhibited the best second-order rate constants for optimal ligand reactivity with the binding site. These analyses provide insights into the relative patterns of covalent inactivation of PAD isozymes and the design of novel inhibitors targeting PAD enzymes as potential therapeutic targets.

Immunological targets in inflammation from the small molecule perspective.

Inflammation and autoimmune disorders have received much greater attention in the recent years due to the elucidation of various molecular mechanisms and the discoveries of various cytokines and other proteins involved in these processes. These discoveries are helping develop novel therapeutics including small molecules and protein therapeutics (biologics) for the treatment of sterile and nonsterile inflammatory disorders. Small molecule drugs have several advantages over protein therapeutics including their affordability for chronic treatments. In this review article, recent successes targeting various inflammatory cytokines and the corresponding receptors such as TLRs, interleukins, p38α as well as recent strategies for developing small molecule antagonists using rational models are discussed.

Structural determinants for the inhibitory ligands of orotidine-5'-monophosphate decarboxylase.

In recent years, orotidine-5'-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to design novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.

Structure-activity relationships of orotidine-5'-monophosphate decarboxylase inhibitors as anticancer agents.

A series of 6-substituted and 5-fluoro-6-substituted uridine derivatives were synthesized and evaluated for their potential as anticancer agents. The designed molecules were synthesized from either fully protected uridine or the corresponding 5-fluorouridine derivatives. The mononucleotide derivatives were used for enzyme inhibition investigations against ODCase. Anticancer activities of all the synthesized derivatives were evaluated using the nucleoside forms of the inhibitors. 5-Fluoro-UMP was a very weak inhibitor of ODCase. 6-Azido-5-fluoro and 5-fluoro-6-iodo derivatives are covalent inhibitors of ODCase, and the active site Lys145 residue covalently binds to the ligand after the elimination of the 6-substitution. Among the synthesized nucleoside derivatives, 6-azido-5-fluoro, 6-amino-5-fluoro, and 6-carbaldehyde-5-fluoro derivatives showed potent anticancer activities in cell-based assays against various leukemia cell lines. On the basis of the overall profile, 6-azido-5-fluoro and 6-amino-5-fluoro uridine derivatives exhibited potential for further investigations.

De novo design of nonpeptidic compounds targeting the interactions between interferon-alpha and its cognate cell surface receptor.

Type 1 interferons (IFN) bind specifically to the corresponding receptor, IFNAR. Agonists and antagonists for IFNAR have potential therapeutic value in the treatment of viral infections and systemic lupus erythematosus, respectively. Specific sequences on the surface of IFN, IFN receptor recognition peptides (IRRPs) mediate the binding and signal transduction when IFN interacts with IFNAR. Structural features of two such IRRPs, IRRP-1 and IRRP-3, were used as templates to design small molecule mimetics. In silico screening was used to identify the molecular structural features mimicking their surface characteristics. A set of 26 compounds were synthesized and their ability to interfere with IFN-IFNAR interactions was investigated. Two compounds exhibited antagonist activity, specifically, blocking IFN-inducible Stat phosphorylation Stat complex-DNA binding. Design principles revealed here pave the way toward a novel series of small molecules as antagonists for IFN-IFNAR interactions.

Structure-activity relationships of C6-uridine derivatives targeting plasmodia orotidine monophosphate decarboxylase.

Malaria, caused by Plasmodia parasites, has re-emerged as a major problem, imposing its fatal effects on human health, especially due to multidrug resistance. In Plasmodia, orotidine 5'-monophosphate decarboxylase (ODCase) is an essential enzyme for the de novo synthesis of uridine 5'-monophosphate. Impairing ODCase in these pathogens is a promising strategy to develop novel classes of therapeutics. Encouraged by our recent discovery that 6-iodo uridine is a potent inhibitor of P. falciparum, we investigated the structure-activity relationships of various C6 derivatives of UMP. 6-Cyano, 6-azido, 6-amino, 6-methyl, 6- N-methylamino, and 6- N, N-dimethylamino derivatives of uridine were evaluated against P. falciparum. The mononucleotides of 6-cyano, 6-azido, 6-amino, and 6-methyl uridine derivatives were studied as inhibitors of plasmodial ODCase. 6-Azidouridine 5'-monophosphate is a potent covalent inhibitor of P. falciparum ODCase. 6-Methyluridine exhibited weak antimalarial activity against P. falciparum 3D7 isolate. 6- N-Methylamino and 6- N, N-dimethylamino uridine derivatives exhibited moderate antimalarial activities.

A potent, covalent inhibitor of orotidine 5'-monophosphate decarboxylase with antimalarial activity.

Orotidine 5'-monophosphate decarboxylase (ODCase) has evolved to catalyze the decarboxylation of orotidine 5'-monophosphate without any covalent intermediates. Active site residues in ODCase are involved in an extensive hydrogen-bonding network. We discovered that 6-iodouridine 5'-monophosphate (6-iodo-UMP) irreversibly inhibits the catalytic activities of ODCases from Methanobacterium thermoautotrophicum and Plasmodium falciparum. Mass spectral analysis of the enzyme-inhibitor complex confirms covalent attachment of the inhibitor to ODCase accompanied by the loss of two protons and the iodo moiety. The X-ray crystal structure (1.6 A resolution) of the complex of the inhibitor and ODCase clearly shows the covalent bond formation with the active site Lys-72 [corrected] residue. 6-Iodo-UMP inhibits ODCase in a time- and concentration-dependent fashion. 6-Iodouridine, the nucleoside form of 6-iodo-UMP, exhibited potent antiplasmodial activity, with IC50s of 4.4 +/- 1.3 microM and 6.2 +/- 0.7 microM against P. falciparum ItG and 3D7 isolates, respectively. 6-Iodouridine 5'-monophosphate is a novel covalent inhibitor of ODCase, and its nucleoside analogue paves the way to a new class of inhibitors against malaria.

Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics.

Inhibitors of orotidine monophosphate decarboxylase (ODCase) have applications in RNA viral, parasitic, and other infectious diseases. ODCase catalyzes the decarboxylation of orotidine monophosphate (OMP), producing uridine monophosphate (UMP). Novel inhibitors 6-amino-UMP and 6-cyano-UMP were designed on the basis of the substructure volumes in the substrate OMP and in an inhibitor of ODCase, barbituric acid monophosphate, BMP. A new enzyme assay method using isothermal titration calorimetry (ITC) was developed to investigate the inhibition kinetics of ODCase. The reaction rates were measured by monitoring the heat generated during the decarboxylation reaction of orotidine monophosphate. Kinetic parameters (k(cat) = 21 s(-1) and KM = 5 microM) and the molar enthalpy (DeltaH(app) = 5 kcal/mol) were determined for the decarboxylation of the substrate by ODCase. Competitive inhibition of the enzyme was observed and the inhibition constants (Ki) were determined to be 12.4 microM and 29 microM for 6-aza-UMP and 6-cyano-UMP, respectively. 6-Amino-UMP was found to be among the potent inhibitors of ODCase, having an inhibition constant of 840 nM. We reveal here the first inhibitors of ODCase designed by the principles of bioisosterism and a novel method of using isothermal calorimetry for enzyme inhibition studies.

An unprecedented twist to ODCase catalytic activity.

Orotidine-5'-monophosphate decarboxylase (ODCase) has evolved to catalyze a decarboxylation reaction, most probably via a carbanion species at the C6 position of orotidine-5'-monophosphate. We reveal an unusual biochemical pathway of conversion of 6-cyano-uridine-5'-monophosphate by ODCase to barbiturate-5'-monophosphate via perhaps an electrophilic center at the C6 position, leading to inhibition. This potential of ODCase is very useful in the design of novel inhibitors.