Serine and threonine kinases may contribute to insulin resistance and the development of type 2 diabetes. To test the potential for members of the mitogen-activated protein (MAP) kinase family to contribute to type 2 diabetes, we examined basal and insulin-stimulated Erk 1/2, JNK, and p38 phosphorylation in adipocytes isolated from healthy and type 2 diabetic individuals. Maximal insulin stimulation increased the phosphorylation of Erk 1/2 and JNK in healthy control subjects but not type 2 diabetic patients. Insulin stimulation did not increase p38 phosphorylation in either healthy control subjects or type 2 diabetic patients. In type 2 diabetic adipocytes, the basal phosphorylation status of these MAP kinases was significantly elevated and was associated with decreased IRS-1 and GLUT4 in these fat cells. To determine whether MAP kinases were involved in the downregulation of IRS-1 and GLUT4 protein levels, selective inhibitors were used to inhibit these MAP kinases in 3T3-L1 adipocytes treated chronically with insulin. Inhibition of Erk 1/2, JNK, or p38 had no effect on insulin-stimulated reduction of IRS-1 protein levels. However, inhibition of the p38 pathway prevented the insulin-stimulated decrease in GLUT4 protein levels. In summary, type 2 diabetes is associated with an increased basal activation of the MAP kinase family. Furthermore, upregulation of the p38 pathway might contribute to the loss of GLUT4 expression observed in adipose tissue from type 2 diabetic patients. Diabetes 52:634 -641, 2003

BackgroundThe 8-aminoquinoline (8AQ) drug primaquine (PQ) is currently the only approved drug effective against the persistent liver stage of the hypnozoite forming strains Plasmodium vivax and Plasmodium ovale as well as Stage V gametocytes of Plasmodium falciparum. To date, several groups have investigated the toxicity observed in the 8AQ class, however, exact mechanisms and/or metabolic species responsible for PQ’s haemotoxic and anti-malarial properties are not fully understood.MethodsIn the present study, the metabolism of PQ was evaluated using in vitro recombinant metabolic enzymes from the cytochrome P450 (CYP) and mono-amine oxidase (MAO) families. Based on this information, metabolite identification experiments were performed using nominal and accurate mass measurements.ResultsRelative activity factor (RAF)-weighted intrinsic clearance values show the relative role of each enzyme to be MAO-A, 2C19, 3A4, and 2D6, with 76.1, 17.0, 5.2, and 1.7% contributions to PQ metabolism, respectively. CYP 2D6 was shown to produce at least six different oxidative metabolites along with demethylations, while MAO-A products derived from the PQ aldehyde, a pre-cursor to carboxy PQ. CYPs 2C19 and 3A4 produced only trace levels of hydroxylated species.ConclusionsAs a result of this work, CYP 2D6 and MAO-A have been implicated as the key enzymes associated with PQ metabolism, and metabolites previously identified as potentially playing a role in efficacy and haemolytic toxicity have been attributed to production via CYP 2D6 mediated pathways.

Participation of p38 mitogen-activated protein kinase (p38) in insulin-induced glucose uptake was suggested using pyridinylimidazole p38 inhibitors (e.g. SB203580). However, the role of p38 in insulin action remains controversial. We further test p38 participation in glucose uptake using a dominant-negative p38 mutant and two novel pharmacological p38 inhibitors related to but different from SB203580. We present the structures and activities of the azaazulene pharmacophores A291077 and A304000. p38 kinase activity was inhibited in vitro by A291077 and A304000 (IC 50 ‫؍‬ 0.6 and 4.7 M). At higher concentrations A291077 but not A304000 inhibited JNK2␣ (IC 50 ‫؍‬ 3.5 M). Pretreatment of 3T3-L1 adipocytes and L6 myotubes expressing GLUT4myc (L6-GLUT4myc myotubes) with A291077, A304000, SB202190, or SB203580 reduced insulin-stimulated glucose uptake by 50 -60%, whereas chemical analogues inert toward p38 were ineffective. Expression of an inducible, dominant-negative p38 mutant in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. GLUT4 translocation to the cell surface, immunodetected on plasma membrane lawns of 3T3-L1 adipocytes or on intact L6-GLUT4myc myotubes, was not altered by chemical or molecular inhibition of p38. We propose that p38 contributes to enhancing GLUT4 activity, thereby increasing glucose uptake. In addition, the azaazulene class of inhibitors described will be useful to decipher cellular actions of p38 and JNK.The p38 mitogen-activated protein kinases (p38), also referred to as stress-activated protein kinases-2, are a family of proline-directed serine/threonine kinases (1, 2). At least four isoforms, the products of different genes, have been cloned and are 60 -70% identical in their amino acid sequence. The most commonly used nomenclature of these isoforms are p38␣ (3, 4), p38␤ (5, 6), p38␥ (7,8), and p38␦ (9, 10). A splice variant of the p38␤, referred to as p38␤2, has also been described (11). Northern blot analysis has shown a wide tissue distribution of these isoforms, although p38␤ and p38␥ are preferentially expressed in skeletal muscle (5, 9). In addition to stressors, members of this family of protein kinases can also be activated by growth factors (12-15).Full activation of p38 by pro-inflammatory cytokines requires phosphorylation of Thr-180 and Tyr-182 found within a TGY tripeptide motif in the activation loop of the kinase (16). This double phosphorylation is catalyzed by the dual-specific MAPK 1 kinases MKK3 and MKK6 and possibly via auto-phosphorylation (17). It is remarkable that stimuli that increase p38 phosphorylation such as insulin-like growth factor-1 (18), muscle contraction (19 -21), lipoic acid (22), 5-aminoimidazole-4-carboxamide ribonucleoside (23), pro-inflammatory cytokines (18), protein synthesis inhibitors (24, 25), hyperosmolar stress (26), and preconditioning (ischemia/reperfusion) (27) also elevate glucose uptake. Importantly, the pyridinylimidazole inhibitor of p38, SB203580, reduced the stimulation of glucose uptake by all of the above stimuli incl...

Primaquine (PQ) metabolism by the cytochrome P450 (CYP) 2D family of enzymes is required for antimalarial activity in both humans (2D6) and mice (2D). Human CYP 2D6 is highly polymorphic, and decreased CYP 2D6 enzyme activity has been linked to decreased PQ antimalarial activity. Despite the importance of CYP 2D metabolism in PQ efficacy, the exact role that these enzymes play in PQ metabolism and pharmacokinetics has not been extensively studied in vivo. In this study, a series of PQ pharmacokinetic experiments were conducted in mice with differential CYP 2D metabolism characteristics, including wild-type (WT), CYP 2D knockout (KO), and humanized CYP 2D6 (KO/knock-in [KO/KI]) mice. Plasma and liver pharmacokinetic profiles from a single PQ dose (20 mg/kg of body weight) differed significantly among the strains for PQ and carboxy-PQ. Additionally, due to the suspected role of phenolic metabolites in PQ efficacy, these were probed using reference standards. Levels of phenolic metabolites were highest in mice capable of metabolizing CYP 2D6 substrates (WT and KO/KI 2D6 mice). PQ phenolic metabolites were present in different quantities in the two strains, illustrating species-specific differences in PQ metabolism between the human and mouse enzymes. Taking the data together, this report furthers understanding of PQ pharmacokinetics in the context of differential CYP 2D metabolism and has important implications for PQ administration in humans with different levels of CYP 2D6 enzyme activity. P rimaquine (PQ) is the only FDA-approved drug for treatment of relapsing infections with malarial strains, including Plasmodium vivax and P. ovale (1-3). PQ belongs to the 8-aminoquinoline (8AQ) class of antimalarial compounds, among which several molecules, including PQ, have potent antihypnozoite activity (2, 4, 5). Low doses of PQ are also recommended for malaria transmission-blocking efforts due to PQ's gametocidal activity (6, 7). PQ's utility in malaria treatment and potential use in malarial transmission reduction and malaria eradication efforts require an understanding of the molecular species involved in its mechanism of action.Recent reports have shown that PQ requires metabolic activation by the cytochrome P450 (CYP) 2D isoenzymes for liver-stage antimalarial activity in both mouse studies (CYP 2D) and human studies (CYP 2D6) (8-11). Pybus et al. demonstrated that PQ was active only in mice capable of metabolizing CYP 2D6 substrates. Deletion of the mouse enzyme closest to human CYP 2D6 (mouse CYP 2D22 via deletion of the CYP 2D gene cluster) in mice completely blocked liver-stage antimalarial activity in vivo (10). The study by Bennett et al. demonstrated a direct link between CYP 2D6 metabolizer status and PQ efficacy for P. vivax treatment in several human subjects (8). PQ therapy is of significant importance for P. vivax radical cure, presumptive antirelapse therapy (PART), and malaria eradication efforts, and the requirement of CYP 2D6 metabolism for PQ efficacy is problematic because CYP 2D6 is highly polymorp...

BackgroundTafenoquine (TQ) is an 8-aminoquinoline (8AQ) that has been tested in several Phase II and Phase III clinical studies and is currently in late stage development as an anti-malarial prophylactic agent. NPC-1161B is a promising 8AQ in late preclinical development. It has recently been reported that the 8AQ drug primaquine requires metabolic activation by CYP 2D6 for efficacy in humans and in mice, highlighting the importance of pharmacogenomics in the target population when administering primaquine. A logical follow-up study was to determine whether CYP 2D activation is required for other compounds in the 8AQ structural class.MethodsIn the present study, the anti-malarial activities of NPC-1161B and TQ were assessed against luciferase expressing Plasmodium berghei in CYP 2D knock-out mice in comparison with normal C57BL/6 mice (WT) and with humanized/CYP 2D6 knock-in mice by monitoring luminescence with an in vivo imaging system. These experiments were designed to determine the direct effects of CYP 2D metabolic activation on the anti-malarial efficacy of NPC-1161B and TQ.ResultsNPC-1161B and TQ exhibited no anti-malarial activity in CYP 2D knock-out mice when dosed at their ED100 values (1 mg/kg and 3 mg/kg, respectively) established in WT mice. TQ anti-malarial activity was partially restored in humanized/CYP 2D6 knock-in mice when tested at two times its ED100.ConclusionsThe results reported here strongly suggest that metabolism of NPC-1161B and TQ by the CYP 2D enzyme class is essential for their anti-malarial activity. Furthermore, these results may provide a possible explanation for therapeutic failures for patients who do not respond to 8AQ treatment for relapsing malaria. Because CYP 2D6 is highly polymorphic, variable expression of this enzyme in humans represents a significant pharmacogenomic liability for 8AQs which require CYP 2D metabolic activation for efficacy, particularly for large-scale prophylaxis and eradication campaigns.

ObjectivesDrugs for Neglected Diseases initiative (DNDi) has identified three chemical lead series, the nitroimidazoles, benzoxaboroles and aminopyrazoles, as innovative treatments for visceral leishmaniasis. The leads discovered using phenotypic screening, were optimised following disease- and compound-specific criteria. Several leads of each series were progressed and preclinical drug candidates have been nominated. Here we evaluate the efficacy of the lead compounds of each of these three chemical classes in in vitro and in vivo models of cutaneous leishmaniasis.MethodsThe in vitro activity of fifty-five compounds was evaluated against the intracellular amastigotes of L. major, L. aethiopica, L. amazonensis, L. panamensis, L. mexicana and L. tropica. The drugs demonstrating potent activity (EC50 < 5 μM) against at least 4 of 6 species were subsequently evaluated in vivo in different L. major – BALB/c mouse models using a 5 or 10-day treatment with either the oral or topical formulations. Efficacy was expressed as lesion size (measured daily using callipers), parasite load (by quantitative PCR – DNA) and bioluminescence signal reduction relative to the untreated controls.ResultsThe selected drug compounds (3 nitroimidazoles, 1 benzoxaborole and 3 aminopyrazoles) showed consistent and potent activity across a range of Leishmania species that are known to cause CL with EC50 values ranging from 0.29 to 18.3 μM. In all cases, this potent in vitro antileishmanial activity translated into high levels of efficacy with a linear dose-response against murine CL. When administered at 50 mg/kg/day, DNDI-0690 (nitroimidazole), DNDI-1047 (aminopyrazole) and DNDI-6148 (benzoxaborole) all resulted in a significant lesion size reduction (no visible nodule) and an approximate 2-log-fold reduction of the parasite load as measured by qPCR compared to the untreated control.ConclusionsThe lead compounds DNDI-0690, DNDI-1047 and DNDI-6148 showed excellent activity across a range of Leishmania species in vitro and against L. major in mice. These compounds offer novel potential drugs for the treatment of CL.

An enantioselective total synthesis of (−)-chlorothricolide (1) has been completed via a route involving the tandem inter- and intramolecular Diels−Alder (IMDA) reaction of hexaenoate 19 and the chiral dienophile (R)-12. This reaction, which establishes seven asymmetric centers in a single operation, is feasible only by virtue of the high diastereofacial and exo selectivity of dienophile 12. The C(9)-trimethylsilyl steric directing group of 19 also plays a key role by controlling the stereochemical course of the IMDA reaction leading to the bottom half octahydronaphthalene unit. Hexaenoate 19 was prepared in 32% overall yield by a 10-step sequence starting from the known acetylenic ketone 33. Key steps include the asymmetric reduction of 33 using Alpine Borane (up to 94% ee), the Suzuki cross coupling of α-iodo vinylsilane 20 with vinylboronic acid 21, and the Horner-type olefination of aldehyde 41 with dienylic phosphonate 22. The key tandem inter-intramolecular Diels−Alder reaction was performed by heating a mixture of 19 and (R)-12 (2 equiv) in toluene at 120 °C, which provided the targeted double cycloadduct 44 in 40−45% yield, along with 19% of other cycloadduct isomers and 25−20% of the IMDA adduct 24 with an (E,E,E)-C(16)−C(21) triene. The latter compound was recycled by treatment with additional (R)-12 in trichloroethylene at 125 °C. The yield of 44 from hexaenoate 19 was 55−59% after one recycle of (E,E,E)-24. Elaboration of 44 to (−)-chlorothricolide was accomplished by a 9-step sequence in 26% overall yield, key steps of which included the construction of the spirotetronate subunit of 51 via the Dieckmann cyclization of 50, deprotection of the two allyl units with Pd(0) catalysis, and the BOP-Cl-mediated macrolactonization of seco acid 52. The vinyl trimethylsilane substituent was removed in the final step of the synthesis by treatment with EtSH and BF3·Et2O. Because an authentic sample of chlorothricolide was not available, synthetic (−)-chlorothricolide was treated with CH2N2 to give 24-O-methyl chlorothricolide methyl ester (59) [[α]25 D −29.3° (c = 0.95, CHCl3), mp 228.5−229 °C; lit.1a [α]D 20 −30° (c = 1, CHCl3), lit.1a mp 230 °C)] which proved identical in all respects (except optical rotation and mp) with an authentic sample of racemic 59 provided by Professor Yoshii.

Tropical protozoal infections are a significant cause of morbidity and mortality worldwide; four in particular (human African trypanosomiasis (HAT), Chagas disease, cutaneous leishmaniasis, and malaria) have an estimated combined burden of over 87 million disability-adjusted life years. New drugs are needed for each of these diseases. Building on the previous identification of NEU-617 (1) as a potent and nontoxic inhibitor of proliferation for the HAT pathogen (Trypanosoma brucei), we have now tested this class of analogs against other protozoal species: T. cruzi (Chagas disease), Leishmania major (cutaneous leishmaniasis), and Plasmodium falciparum (malaria). Based on hits identified in this screening campaign, we describe the preparation of several replacements for the quinazoline scaffold and report these inhibitors’ biological activities against these parasites. In doing this, we have identified several potent proliferation inhibitors for each pathogen, such as 4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-6-(4-((4-methyl-1,4-diazepan-1-yl)sulfonyl)phenyl)quinoline-3-carbonitrile (NEU-924, 83) for T. cruzi and N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-7-(4-((4-methyl-1,4-diazepan-1-yl)sulfonyl)phenyl)cinnolin-4-amine (NEU-1017, 68) for L. major and P. falciparum.