Effects of JWH015 in cytokine secretion in primary human keratinocytes and fibroblasts and its suitability for topical/transdermal delivery.

Background JWH015 is a cannabinoid (CB) receptor type 2 agonist that produces immunomodulatory effects. Since skin cells play a key role in inflammatory conditions and tissue repair, we investigated the ability of JWH015 to promote an anti-inflammatory and pro-wound healing phenotype in human primary skin cells. Methods Human primary keratinocytes and fibroblasts were stimulated with lipopolysaccharide. The mRNA expression of cannabinoid receptors was determined using RT-PCR. The effects of JWH015 (0.05, 0.1, 0.5, and 1 µM) in pro- and anti-inflammatory factors were tested in lipopolysaccharide-stimulated cells. A scratch assay, using a co-culture of keratinocytes and fibroblasts, was used to test the effects of JWH015 in wound healing. In addition, the topical and transdermal penetration of JWH015 was studied in Franz diffusion cells using porcine skin and LC-MS. Results The expression of CB1 and CB2 receptors (mRNA) and the production of pro- and anti-inflammatory factors enhanced in keratinocytes and fibroblasts following lipopolysaccharide stimulation. JWH015 reduced the concentration of major pro-inflammatory factors (IL-6 and MCP-1) and increased the concentration of a major anti-inflammatory factor (TGF-ß) in lipopolysaccharide-stimulated cells. JWH015 induced a faster scratch gap closure. These JWH015'seffects were mainly modulated through both CB1 and CB2 receptors. Topically administered JWH015 was mostly retained in the skin and displayed a sustained and low level of transdermal permeation. Conclusions Our findings suggest that targeting keratinocytes and fibroblasts with cannabinoid drugs could represent a therapeutic strategy to resolve peripheral inflammation and promote tissue repair.

Synthesis of bi-substrate state mimics of dihydropteroate synthase as potential inhibitors and molecular probes.

The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillusanthracis dihydropteroate synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of dihydropteroate synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the dihydropteroate synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics.

Diastereoselective synthesis of a spironoraristeromycin using an acylnitroso Diels-Alder reaction.

The tert-butyl N-hydroxycarbamate-derived nitroso reagent 1 reacted with N-Cbz-protected spirocyclic diene 2 to provide spirocycloadduct 3. Here we describe the efficient conversion of 3 into the novel carbocyclic nucleoside spironoraristeromycin 4.

Recent Advances in Drug Repurposing for Parkinson's Disease.

As a long-term degenerative disorder of the central nervous system that mostly affects older people, Parkinson's disease is a growing health threat to our ever-aging population. Despite remarkable advances in our understanding of this disease, all therapeutics currently available only act to improve symptoms but cannot stop the disease progression. Therefore, it is essential that more effective drug discovery methods and approaches are developed, validated, and used for the discovery of disease-modifying treatments for Parkinson's disease. Drug repurposing, also known as drug repositioning, or the process of finding new uses for existing or abandoned pharmaceuticals, has been recognized as a cost-effective and timeefficient way to develop new drugs, being equally promising as de novo drug discovery in the field of neurodegeneration and, more specifically for Parkinson's disease. The availability of several established libraries of clinical drugs and fast evolvement in disease biology, genomics and bioinformatics has stimulated the momentums of both in silico and activity-based drug repurposing. With the successful clinical introduction of several repurposed drugs for Parkinson's disease, drug repurposing has now become a robust alternative approach to the discovery and development of novel drugs for this disease. In this review, recent advances in drug repurposing for Parkinson's disease will be discussed.

Chemical knockout of pantothenate kinase reveals the metabolic and genetic program responsible for hepatic coenzyme A homeostasis.

Coenzyme A (CoA) is the major acyl group carrier in intermediary metabolism. Hopantenate (HoPan), a competitive inhibitor of the pantothenate kinases, was used to chemically antagonize CoA biosynthesis. HoPan dramatically reduced liver CoA and mice developed severe hypoglycemia. Insulin was reduced, glucagon and corticosterone were elevated, and fasting accelerated hypoglycemia. Metabolic profiling revealed a large increase in acylcarnitines, illustrating the role of carnitine in buffering acyl groups to maintain the nonesterified CoASH level. HoPan triggered significant changes in hepatic gene expression that substantially increased the thioesterases, which liberate CoASH from acyl-CoA, and increased pyruvate dehydrogenase kinase 1, which prevents the conversion of CoASH to acetyl-CoA. These results identify the metabolic rearrangements that maintain the CoASH pool which is critical to mitochondrial functions, including gluconeogenesis, fatty acid oxidation, and the tricarboxylic acid and urea cycles.

Development of an etoposide prodrug for dual prodrug-enzyme antitumor therapy.

Enzyme-prodrug approaches to cancer therapy, theoretically, have the potential to mediate tumor-selective cytotoxicity. However, even if tumor-specific prodrug activation is achieved, enzyme-prodrug systems investigated thus far comprised a single enzyme and a specific prodrug. Although targeted, such systems constitute single-agent therapy, which may be ineffective and/or may promote development of drug resistance. Therefore, a goal of our laboratories was to design and characterize a novel dipiperidinyl derivative of etoposide [1,4'-dipiperidine-1'-carboxylate-etoposide (dp-VP16)] that would act as a prodrug. We envisioned that dp-VP16 would be converted to the active chemotherapeutic agent VP-16 by the same rabbit carboxylesterase (rCE) that we have previously shown to efficiently activate the prodrug irinotecan (CPT-11). This dp-VP16 prodrug might then be used in combination with CPT-11, with both drugs activated by a single enzyme. We evaluated the ability of pure rCE and two human carboxylesterases, hCE1 and hiCE (hCE2), to activate dp-VP16 in vitro, and in neuroblastoma cell lines designed to express/overexpress each enzyme. In SK-N-AS neuroblastoma cell transfectants, expression of rCE or hiCE decreased the IC50 of dp-VP16 as a single agent by 8.3- and 3.4-fold, respectively, in growth inhibition assays. Purified hCE1 did not metabolize dp-VP16 in vitro and did not affect its IC50 in intact cells. The combination indices of sequential exposure to CPT-11 followed by dp-VP16 ranged from approximately 0.4 to 0.6, suggesting that this combination produced greater-than-additive cytotoxicity in neuroblastoma cells expressing rCE. These data provide proof-of-principle that enzyme-prodrug therapy approaches comprised of prodrugs with complementary mechanisms of cytotoxicity that are activated by a single enzyme can be developed.

Recent Advances in the Rational Design and Optimization of Antibacterial Agents.

This review discusses next-generation antibacterial agents developed using rational, or targeted, drug design strategies. The focus of this review is on small-molecule compounds that have been designed to bypass developing bacterial resistance, improve the antibacterial spectrum of activity, and/or to optimize other properties, including physicochemical and pharmacokinetic properties. Agents are discussed that affect known antibacterial targets, such as the bacterial ribosome, nucleic acid binding proteins, and proteins involved in cell-wall biosynthesis; as well as some affecting novel bacterial targets which do not have currently marketed agents. The discussion of the agents focuses on the rational design strategies employed and the synthetic medicinal chemistry and structure-based design techniques utilized by the scientists involved in the discoveries, including such methods as ligand- and structure-based strategies, structure-activity relationship (SAR) expansion strategies, and novel synthetic organic chemistry methods. As such, the discussion is limited to small-molecule therapeutics that have confirmed macromolecular targets and encompasses only a fraction of all antibacterial agents recently approved or in late-stage clinical trials. The antibacterial agents selected have been recently approved for use on the U.S. or European markets or have shown promising results in phase 2 or phase 3 U.S.

Pantothenamides are potent, on-target inhibitors of Plasmodium falciparum growth when serum pantetheinase is inactivated.

Growth of the virulent human malaria parasite Plasmodium falciparum is dependent on an extracellular supply of pantothenate (vitamin B(5)) and is susceptible to inhibition by pantothenate analogues that hinder pantothenate utilization. In this study, on the hunt for pantothenate analogues with increased potency relative to those reported previously, we screened a series of pantothenamides (amide analogues of pantothenate) against P. falciparum and show for the first time that analogues of this type possess antiplasmodial activity. Although the active pantothenamides in this series exhibit only modest potency under standard in vitro culture conditions, we show that the potency of pantothenamides is selectively enhanced when the parasite culture medium is pre-incubated at 37°C for a prolonged period. We present evidence that this finding is linked to the presence in Albumax II (a serum-substitute routinely used for in vitro cultivation of P. falciparum) of pantetheinase activity: the activity of an enzyme that hydrolyzes the pantothenate metabolite pantetheine, for which pantothenamides also serve as substrates. Pantetheinase activity, and thereby pantothenamide degradation, is reduced following incubation of Albumax II-containing culture medium for a prolonged period at 37°C, revealing the true, sub-micromolar potency of pantothenamides. Importantly we show that the potent antiplasmodial effect of pantothenamides is attenuated with pantothenate, consistent with the compounds inhibiting parasite proliferation specifically by inhibiting pantothenate and/or CoA utilization. Additionally, we show that the pantothenamides interact with P. falciparum pantothenate kinase, the first enzyme involved in converting pantothenate to coenzyme A. This is the first demonstration of on-target antiplasmodial pantothenate analogues with sub-micromolar potency, and highlights the potential of pantetheinase-resistant pantothenamides as antimalarial agents.

Structure-activity relationships and enzyme inhibition of pantothenamide-type pantothenate kinase inhibitors.

A set of novel pantothenamide-type analogues of the known Staphylococcus aureus pantothenate kinase (SaPanK) inhibitors, N-pentyl, and N-heptylpantothenamide, was synthesized in three series. The first series of analogues (1-3) were designed as molecular probes of the PanK binding site to elucidate important structure-activity relationships (SAR). The second series of analogues (4-16) were designed using structural information obtained from the Escherichia coli PanK (EcPanK) structure by targeting the pantothenate binding site and the adjacent phenylalanine-lined lipophilic pocket. Insight into the antimicrobial effect of N-pentylpantothenamide (N5-Pan) through its conversion to the antimetabolite ethyldethia-CoA and further incorporation into an inactive acyl carrier protein analogue drove the development of the third series of analogues (17-25) to enhance this effect using substrate-like substitutions. Each of the analogues was screened for enzyme inhibition activity against a panel of pantothenate kinases consisting of EcPanK, Aspergillus nidulans (AnPanK), SaPanK, and the murine isoform (MmPanK1alpha). Series 1 demonstrated only modest inhibitory activity, but did reveal some important SAR findings including stereospecific binding. Series 2 demonstrated a much higher inhibition rate for the entire series and significant inhibition was seen with analogues containing alkyl substituents. Series 3 demonstrated the most preferential inhibition profile, with the highest inhibitory activity against the SaPanK and MmPanK1alpha. The MmPanK1alpha protein was inhibited by a broad spectrum of the compounds, whereas the E. coli enzyme showed greater selectivity. The overall activity data from these analogues suggest a complex and non-enzyme specific SAR for pantothenamide substrate/inhibitors of the different PanK enzymes.

A pantothenate kinase from Staphylococcus aureus refractory to feedback regulation by coenzyme A.

The key regulatory step in CoA biosynthesis in bacteria and mammals is pantothenate kinase (CoaA), which governs the intracellular concentration of CoA through feedback regulation by CoA and its thioesters. CoaA from Staphylococcus aureus (SaCoaA) has a distinct primary sequence that is more similar to the mammalian pantothenate kinases than the prototypical bacterial CoaA of Escherichia coli. In contrast to all known pantothenate kinases, SaCoaA activity is not feedback-regulated by CoA or CoA thioesters. Metabolic labeling of S. aureus confirms that CoA levels are not controlled by CoaA or at steps downstream from CoaA. The pantothenic acid antimetabolite N-heptylpantothenamide (N7-Pan) possesses potent antimicrobial activity against S. aureus and has multiple cellular targets. N7-Pan is a substrate for SaCoaA and is converted to the inactive butyldethia-CoA analog by the downstream pathway enzymes. The analog is also incorporated into acyl carrier protein and D-alanyl carrier protein, the prosthetic groups of which are derived from CoA. The inactivation of acyl carrier protein and the cessation of fatty acid synthesis are the most critical causes of growth inhibition by N7-Pan because the toxicity of the drug is ameliorated by supplementing the growth medium with fatty acids. The absence of feedback regulation at the pantothenate kinase step allows the accumulation of high concentrations of intracellular CoA, consistent with the physiology of S. aureus, which lacks glutathione and relies on the CoA/CoA disulfide reductase redox system for protection from oxidative damage.

Discovery of non-carbohydrate inhibitors of aminoglycoside-modifying enzymes.

Chemical modification and inactivation of aminoglycosides by many different enzymes expressed in pathogenic bacteria are the main mechanisms of bacterial resistance to these antibiotics. In this work, we designed inhibitors that contain the 1,3-diamine pharmacophore shared by all aminoglycoside antibiotics that contain the 2-deoxystreptamine ring. A discovery library of molecules was prepared by attaching different side chains to both sides of the 1,3-diamine motif. Several of these diamines showed inhibitory activity toward two or three different representative aminoglycoside-modifying enzymes (AGMEs). These studies yielded the first non-carbohydrate inhibitor N-cyclohexyl-N'-(3-dimethylamino-propyl)-propane-1,3-diamine (Compound G,H) that is competitive with respect to the aminoglycoside binding to the enzyme aminoglycoside-2''-nucleotidyltransferase-Ia (ANT2''). Another diamine molecule N-[2-(3,4-dimethoxyphenyl)-ethyl]-N'-(3-dimethylamino-propyl)-propane-1,3-diamine (Compound H,I) was shown to be a competitive inhibitor of two separate enzymes (aminoglycoside-3'-phosphotransferase-IIIa (APH3') and ANT2'') with respect to metal-ATP. Thermodynamic and structural-binding properties of the complexes of APH3' with substrates and inhibitor were shown to be similar to each other, as determined by isothermal titration calorimetry and NMR spectroscopy.

Acyl carrier protein is a cellular target for the antibacterial action of the pantothenamide class of pantothenate antimetabolites.

Pantothenate is the precursor of the essential cofactor coenzyme A (CoA). Pantothenate kinase (CoaA) catalyzes the first and regulatory step in the CoA biosynthetic pathway. The pantothenate analogs N-pentylpantothenamide and N-heptylpantothenamide possess antibiotic activity against Escherichia coli. Both compounds are substrates for E. coli CoaA and competitively inhibit the phosphorylation of pantothenate. The phosphorylated pantothenamides are further converted to CoA analogs, which were previously predicted to act as inhibitors of CoA-dependent enzymes. Here we show that the mechanism for the toxicity of the pantothenamides is due to the inhibition of fatty acid biosynthesis through the formation and accumulation of the inactive acyl carrier protein (ACP), which was easily observed as a faster migrating protein using conformationally sensitive gel electrophoresis. E. coli treated with the pantothenamides lost the ability to incorporate [1-(14)C]acetate to its membrane lipids, indicative of the inhibition of fatty acid synthesis. Cellular CoA was maintained at the level sufficient for bacterial protein synthesis. Electrospray ionization time-of-flight mass spectrometry confirmed that the inactive ACP was the product of the transfer of the inactive phosphopantothenamide moiety of the CoA analog to apo-ACP, forming the ACP analog that lacks the sulfhydryl group for the attachment of acyl chains for fatty acid synthesis. Inactive ACP accumulated in pantothenamide-treated cells because of the active hydrolysis of regular ACP and the slow turnover of the inactive prosthetic group. Thus, the pantothenamides are pro-antibiotics that inhibit fatty acid synthesis and bacterial growth because of the covalent modification of ACP.

The structure of the pantothenate kinase.ADP.pantothenate ternary complex reveals the relationship between the binding sites for substrate, allosteric regulator, and antimetabolites.

Pantothenate kinase catalyzes the first step in the biosynthesis of coenzyme A, the major acyl group carrier in biology. In bacteria, regulation of pantothenate kinase activity is a major factor in controlling intracellular coenzyme A levels, and pantothenate analogs are growth-inhibiting antimetabolites. We have extended the structural information on Escherichia coli pantothenate kinase by determining the structure of the enzyme.ADP. pantothenate ternary complex. Pantothenate binding induces a significant conformational change in amino acids 243-263, which form a "lid" that folds over the open pantothenate binding groove. The positioning of the substrates suggests the reaction proceeds by a concerted mechanism that involves a dissociative transition state, although the negative charge neutralization of the gamma-phosphate by Arg-243, Lys-101, and Mg(2+) coupled with hydrogen bonding of the C1 of pantothenate to Asp-127 suggests different interpretations of the phosphoryl transfer mechanism of pantothenate kinase. N-alkylpantothenamides are substrates for pantothenate kinase. Modeling these antimetabolites into the pantothenate active site predicts that they bind in the same orientation as pantothenate with their alkyl chains interacting with the hydrophobic dome over the pantothenate pocket, which is also accessed by the beta-mercaptoethylamine moiety of the allosteric regulator, coenzyme A. These structural/biochemical studies illustrate the intimate relationship between the substrate, allosteric regulator, and antimetabolite binding sites on pantothenate kinase and provide a framework for studies of its catalysis and feedback regulation.