Synthesis and antileishmanial evaluation of thiazole orange analogs.

Cyanine compounds have previously shown excellent in vitro and promising in vivo antileishmanial efficacy, but the potential toxicity of these agents is a concern. A series of 22 analogs of thiazole orange ((Z)-1-methyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium salt), a commercial cyanine dye with antileishmanial activity, were synthesized in an effort to increase the selectivity of such compounds while maintaining efficacy. Cyanines possessing substitutions on the quinolinium ring system displayed potency against Leishmania donovani axenic amastigotes that differed little from the parent compound (IC50 12-42 nM), while ring disjunction analogs were both less potent and less toxic. Changes in DNA melting temperature were modest when synthetic oligonucleotides were incubated with selected analogs (ΔTm ≤ 5 °C), with ring disjunction analogs showing the least effect on this parameter. Despite the high antileishmanial potency of the target compounds, their toxicity and relatively flat SAR suggests that further information regarding the target(s) of these molecules is needed to aid their development as antileishmanials.

Microneedle-Based Delivery of Amphotericin B for Treatment of Cutaneous Leishmaniasis.

Current therapeutic options against cutaneous leishmaniasis are plagued by several weaknesses. The effective topical delivery of an antileishmanial drug would be useful in treating some forms of cutaneous leishmaniasis. Toward this end, a microneedle based delivery approach for the antileishmanial drug amphotericin B was investigated in murine models of both New World (Leishmania mexicana) and Old World (Leishmania major) infection. In the L. mexicana model, ten days of treatment began on day 35 post infection, when the area of nodules averaged 9-15 mm2. By the end of the experiment, a significant difference in nodule area was observed for all groups receiving topical amphotericin B at 25 mg/kg/day after application of microneedle arrays of 500, 750, and 1000 µM in nominal length compared to the group that received this dose of topical amphotericin B alone. In the L. major model, ten days of treatment began on day 21 post infection when nodule area averaged 51-65 mm2 in the groups. By the end of the experiment, there was no difference in nodule area between the group receiving 25 mg/kg of topical amphotericin B after microneedle application and any of the non-AmBisome groups. These results show the promise of topical delivery of amphotericin B via microneedles in treating relatively small nodules caused by L. mexicana. These data also show the limitations of the approach against a disseminated L. major infection. Further optimization of microneedle delivery is needed to fully exploit this strategy for cutaneous leishmaniasis treatment.

Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations.

Given the limitations of current antileishmanial drugs and the utility of oral combination therapy for other infections, developing an oral combination against visceral leishmaniasis should be a high priority. In vitro combination studies with DB766 and antifungal azoles against intracellular Leishmania donovani showed that posaconazole and ketoconazole, but not fluconazole, enhanced DB766 potency. Pharmacokinetic analysis of DB766-azole combinations in uninfected Swiss Webster mice revealed that DB766 exposure was increased by higher posaconazole and ketoconazole doses, while DB766 decreased ketoconazole exposure. In L. donovani-infected BALB/c mice, DB766-posaconazole combinations given orally for 5 days were more effective than DB766 or posaconazole alone. For example, 81% ± 1% (means ± standard errors) inhibition of liver parasite burden was observed for 37.5 mg/kg of body weight DB766 plus 15 mg/kg posaconazole, while 37.5 mg/kg DB766 and 15 mg/kg posaconazole administered as monotherapy gave 40% ± 5% and 21% ± 3% inhibition, respectively. Combination index (CI) analysis indicated that synergy or moderate synergy was observed in six of nine combined dose groups, while the other three were nearly additive. Liver concentrations of DB766 and posaconazole increased in almost all combination groups compared to monotherapy groups, although many increases were not statistically significant. For DB766-ketoconazole combinations evaluated in this model, two were antagonistic, one displayed synergy, and one was nearly additive. These data indicate that the efficacy of DB766-posaconazole and DB766-ketoconazole combinations in vivo is influenced in part by the pharmacokinetics of the combination, and that the former combination deserves further consideration in developing new treatment strategies against visceral leishmaniasis.

Synthesis and pharmacological evaluation of mono-arylimidamides as antileishmanial agents.

Arylimidamide (AIA) compounds containing two pyridylimidamide terminal groups (bis-AIAs) possess outstanding in vitro antileishmanial activity, and the frontrunner bis-AIA DB766 (2,5-bis[2-(2-isopropoxy)-4-(2-pyridylimino)aminophenyl]furan) is active in visceral leishmaniasis models when given orally. Eighteen compounds containing a single pyridylimidamide terminal group (mono-AIAs) were synthesized and evaluated for their antileishmanial potential. Six of these compounds exhibited sub-micromolar potency against both intracellular Leishmania donovani and Leishmania amazonensis amastigotes, and three of these compounds also displayed selectivity indexes of 25 or greater for the parasites compared to a J774 macrophage cell line. When given orally at a dose of 100mg/kg/day for five days, compound 1b (N-(3-isopropoxy-4-(5-phenylfuran-2-yl)phenyl)picolinimidamide methanesulfonate) reduced liver parasitemia by 46% in L. donovani-infected mice. Mono-AIAs are thus a new class of candidate molecules for antileishmanial drug development.

Efficacy of ceftazidime in a murine model following a lethal aerosol exposure to Burkholderia pseudomallei.

Melioidosis is an endemic disease in numerous tropical regions. Additionally, the bacterium that causes melioidosis, Burkholderia pseudomallei, has potential to be used as a biological weapon. Therefore, development of effective and affordable medical countermeasures to serve regions affected by the disease and to have medical countermeasures available in the event of a bioterrorism attack remains critical. The current study evaluated the efficacy of eight distinct acute phase ceftazidime treatment regimens administered therapeutically in the murine model. At the conclusion of the treatment period, survival rates were significantly greater in several of the treated groups when compared to the control group. Pharmacokinetics of a single dose of ceftazidime were examined at 150 mg/kg, 300 mg/kg, and 600 mg/kg and were compared to an intravenous clinical dose administered at 2000 mg every eight hours. The clinical dose has an estimated 100% fT > 4*MIC which exceeded the highest murine dose of 300 mg/kg every six hours at 87.2% fT > 4*MIC. Based upon survival at the end of the treatment regimen and supplemented by pharmacokinetic modeling, a daily dose of 1200 mg/kg of ceftazidime, administered every 6 h at 300 mg/kg, provides protection in the acute phase of inhalation melioidosis in the murine model.

Preparation of Nanosensors for Detecting the Activity of Glycosaminoglycan Cleaving Enzymes.

Glycosaminoglycans (GAGs) play crucial roles in several biological processes including cell division, angiogenesis, anticoagulation, neurogenesis, axon guidance and growth, and viral and bacterial infections among others. The GAG cleaving hydrolases/lyases play a major role in the control of GAG structures, functions, and turn over. Dysregulation of GAG cleaving enzymes in vivo are linked to a number of human diseases including cancer, diabetes, atherosclerosis, arthritis, inflammation, and cardiovascular diseases. Several GAG cleaving enzymes are widely used for studying GAG glycobiology: heparitinases, chondroitinases, heparanases, hyaluronidases, and keratanases. Herein, we describe a method to synthesize four distinct nanometal surface energy transfer (NSET)-based gold-GAG-dye conjugates (nanosensors). Heparin, chondroitin sulfate, heparan sulfate, and hyaluronic acid are covalently linked with distinct fluorescent dyes and then immobilized on gold nanoparticles (AuNPs) to build nanosensors that serve as excellent substrates for GAG cleaving enzymes. Upon treatment of nanosensors with their respective GAG cleaving enzymes, dye-labeled oligosaccharides/disaccharides are released from AuNPs resulting in enhanced fluorescence recovery. These nanosensors have a great promise as diagnostic tools in various human pathophysiological conditions for detecting dysregulated expression of GAG cleaving enzymes and also as a sensitive analytical tool for assessing the quality control of pharmaceutical grade heparin polysaccharides that are produced in millions of small- and medium-sized animal slaughter houses worldwide.

Synthesis and Antileishmanial Evaluation of Arylimidamide-Azole Hybrids Containing a Phenoxyalkyl Linker.

Due to the limitations of existing medications, there is a critical need for new drugs to treat visceral leishmaniasis. Since arylimidamides and antifungal azoles both show oral activity in murine visceral leishmaniasis models, a molecular hybridization approach was employed where arylimidamide and azole groups were separated by phenoxyalkyl linkers in an attempt to capitalize on the favorable antileishmanial properties of both series. Among the target compounds synthesized, a greater antileishmanial potency against intracellular Leishmania donovani was observed as the linker length increased from two to eight carbons and when an imidazole ring was employed as the terminal group compared to a 1,2,4-triazole group. Compound 24c (N-(4-((8-(1H-imidazol-1-yl)octyl)oxy)-2-isopropoxyphenyl) picolinimidamide) displayed activity against L. donovani intracellular amastigotes with an IC50 value of 0.53 µM. When tested in a murine visceral leishmaniasis model, compound 24c at a dose of 75 mg/kg/day p.o. for five consecutive days resulted in a modest 33% decrease in liver parasitemia compared to the control group, indicating that further optimization of these molecules is needed. While potent hybrid compounds bearing an imidazole terminal group were also strong inhibitors of recombinant CYP51 from L. donovani, as assessed by a fluorescence-based assay, additional targets are likely to play an important role in the antileishmanial action of these compounds.

Visualizing antithrombin-binding 3-O-sulfated heparan sulfate motifs on cell surfaces.

To map the cellular topography of the rare 3-O-sulfated structural motif of heparan sulfate (HS), we constructed quantum dot-based probes for antithrombin and FGF2, which reveal widely different distribution of the targeted HS motifs. The technology helps show that old and young aortic endothelia display widely different levels of the antithrombin-binding 3-O-sulfated HS motif.

Enzymatic synthesis of heparan sulfate and heparin.

Heparan sulfate (HS) polysaccharide chains have been shown to orchestrate distinct biological functions in several systems. Study of HS structure-function relations is, however, hampered due to the lack of availability of HS in sufficient quantities as well as the molecular heterogeneity of naturally occurring HS. Enzymatic synthesis of HS is an attractive alternative to the use of naturally occurring HS, as it reduces molecular heterogeneity, or a long and daunting chemical synthesis of HS. Heparosan, produced by E. coli K5 bacteria, has a structure similar to the unmodified HS backbone structure and can be used as a precursor in the enzymatic synthesis of HS-like polysaccharides. Here, we describe an enzymatic approach to synthesize several specifically sulfated HS polysaccharides for biological studies using the heparosan backbone and a combination of recombinant biosynthetic enzymes such as C5-epimerase and sulfotransferases.

Chemoenzymatically prepared heparan sulfate containing rare 2-O-sulfonated glucuronic acid residues.

The structural diversity of natural sulfated glycosaminoglycans (GAGs) presents major promise for discovery of chemical biology tools or therapeutic agents. Yet, few GAGs have been identified so far to exhibit this promise. We reasoned that a simple approach to identify such GAGs is to explore sequences containing rare residues, for example, 2-O-sulfonated glucuronic acid (GlcAp2S). Genetic algorithm-based computational docking and filtering suggested that GlcAp2S containing heparan sulfate (HS) may exhibit highly selective recognition of antithrombin, a key plasma clot regulator. HS containing only GlcAp2S and 2-N-sulfonated glucosamine residues, labeled as HS2S2S, was chemoenzymatically synthesized in just two steps and was found to preferentially bind antithrombin over heparin cofactor II, a closely related serpin. Likewise, HS2S2S directly inhibited thrombin but not factor Xa, a closely related protease. The results show that a HS containing rare GlcAp2S residues exhibits the unusual property of selective antithrombin activation and direct thrombin inhibition. More importantly, HS2S2S is also the first molecule to activate antithrombin nearly as well as the heparin pentasaccharide although being completely devoid of the critical 3-O-sulfonate group. Thus, this work shows that novel functions and mechanisms may be uncovered by studying rare GAG residues/sequences.

Exploring the mode of action of ebselen in Trypanosoma brucei hexokinase inhibition.

Glycolysis is essential to Trypanosoma brucei, the causative agent of African sleeping sickness, suggesting enzymes in the pathway could be targets for drug development. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one, EbSe) was identified in a screen as a potent inhibitor of T. brucei hexokinase 1 (TbHK1), the first enzyme in the pathway. EbSe has a history of promiscuity as an enzyme inhibitor, inactivating proteins through seleno-sulfide conjugation with Cys residues. Indeed, dilution of TbHK1 and inhibitor following incubation did not temper inhibition suggesting conjugate formation. Using mass spectrometry to analyze EbSe-based modifications revealed that two Cys residues (C327 and C369) were oxidized after treatment. Site-directed mutagenesis of C327 led to enzyme inactivation indicating that C327 was essential for catalysis. C369 was not essential, suggesting that EbSe inhibition of TbHK1 was the consequence of modification of C327 via thiol oxidation. Additionally, neither EbSe treatment nor mutation of the nine TbHK1 Cys residues appreciably altered enzyme quaternary structure.

Extra-glycosomal localisation of Trypanosoma brucei hexokinase 2.

The majority of the glycolytic enzymes in the African trypanosome are compartmentalised within peroxisome-like organelles, the glycosomes. Polypeptides harbouring peroxisomal targeting sequences (PTS type 1 or 2) are targeted to these organelles. This targeting is essential to parasite viability, as compartmentalisation of glycolytic enzymes prevents unregulated ATP-dependent phosphorylation of intermediate metabolites. Here, we report the surprising extra-glycosomal localisation of a PTS-2 bearing trypanosomal hexokinase, TbHK2. In bloodstream form parasites, the protein localises to both glycosomes and to the flagellum. Evidence for this includes fractionation and immunofluorescence studies using antisera generated against the authentic protein as well as detection of epitope-tagged recombinant versions of the protein. In the insect stage parasite, distribution is different, with the polypeptide localised to glycosomes and proximal to the basal bodies. The function of the extra-glycosomal protein remains unclear. While its association with the basal body suggests that it may have a role in locomotion in the insect stage parasite, no detectable defect in directional motility or velocity of cell movement were observed for TbHK2-deficient cells, suggesting that the protein may have a different function in the cell.