Treatment of Organophosphorus Poisoning with 6-Alkoxypyridin-3-ol Quinone Methide Precursors: Resurrection of Methylphosphonate-Aged Acetylcholinesterase.

Organophosphorus (OP) nerve agents inhibit acetylcholinesterase (AChE), creating a cholinergic crisis in which death can occur. The phosphylated serine residue spontaneously dealkylates to the OP-aged form, which current therapeutics cannot reverse. Soman's aging half-life is 4.2 min, so immediate recovery (resurrection) of OP-aged AChE is needed. In 2018, we showed pyridin-3-ol-based quinone methide precursors (QMPs) can resurrect OP-aged electric eel AChE in vitro, achieving 2% resurrection after 24 h of incubation (pH 7, 4 mM). We prepared 50 unique 6-alkoxypyridin-3-ol QMPs with 10 alkoxy groups and five amine leaving groups to improve AChE resurrection. These compounds are predicted in silico to cross the blood-brain barrier and treat AChE in the central nervous system. This library resurrected 7.9% activity of OP-aged recombinant human AChE after 24 h at 250 µM, a 4-fold increase from our 2018 report. The best QMP (1b), with a 6-methoxypyridin-3-ol core and a diethylamine leaving group, recovered 20.8% (1 mM), 34% (4 mM), and 42.5% (predicted maximum) of methylphosphonate-aged AChE activity over 24 h. Seven QMPs recovered activity from AChE aged with Soman and a VX degradation product (EA-2192). We hypothesize that QMPs form the quinone methide (QM) to realkylate the phosphylated serine residue as the first step of resurrection. We calculated thermodynamic energetics for QM formation, but there was no trend with the experimental biochemical data. Molecular docking studies revealed that QMP binding to OP-aged AChE is not the determining factor for the observed biochemical trends; thus, QM formation may be enzyme-mediated.

4-Amidophenol Quinone Methide Precursors: Effective and Broad-Scope Nonoxime Reactivators of Organophosphorus-Inhibited Cholinesterases and Resurrectors of Organophosphorus-Aged Acetylcholinesterase.

Acetylcholinesterase (AChE) inhibition by organophosphorus (OP) compounds poses a serious health risk to humans. While many therapeutics have been tested for treatment after OP exposure, there is still a need for efficient reactivation against all kinds of OP compounds, and current oxime therapeutics have poor blood-brain barrier penetration into the central nervous system, while offering no recovery in activity from the OP-aged forms of AChE. Herein, we report a novel library of 4-amidophenol quinone methide precursors (QMP) that provide effective reactivation against multiple OP-inhibited forms of AChE in addition to resurrecting the aged form of AChE after exposure to a pesticide or some phosphoramidates. Furthermore, these QMP compounds also reactivate OP-inhibited butyrylcholinesterase (BChE) which is an in vivo, endogenous scavenger of OP compounds. The in vitro efficacies of these QMP compounds were tested for reactivation and resurrection of soluble forms of human AChE and BChE and for reactivation of cholinesterases within human blood as well as blood and brain samples from a humanized mouse model. We identify compound 10c as a lead candidate due to its broad-scope efficacy against multiple OP compounds as well as both cholinesterases. With methylphosphonates, compound 10c (250 µM, 1 h) shows >60% recovered activity from OEt-inhibited AChE in human blood as well as mouse blood and brain, thus highlighting its potential for future in vivo analysis. For 10c, the effective concentration (EC50) is less than 25 µM for reactivation of three different methylphosphonate-inhibited forms of AChE, with a maximum reactivation yield above 80%. Similarly, for OP-inhibited BChE, 10c has EC50 values that are less than 150 µM for two different methylphosphonate compounds. Furthermore, an in vitro kinetic analysis show that 10c has a 2.2- and 92.1-fold superior reactivation efficiency against OEt-inhibited and OiBu-inhibited AChE, respectively, when compared to an oxime control. In addition to 10c being a potent reactivator of AChE and BChE, we also show that 10c is capable of resurrecting (ethyl paraoxon)-aged AChE, which is another current limitation of oximes.

In-silico guided design, screening, and molecular dynamic simulation studies for the identification of potential SARS-CoV-2 main protease inhibitors for the targeted treatment of COVID-19.

COVID-19, the disease responsible for the recent pandemic, is caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main protease (Mpro) of SARS-CoV-2 is an essential proteolytic enzyme that plays a number of important roles in the replication of the virus in human host cells. Blocking the function of SARS-CoV-2 Mpro offers a promising and targeted, therapeutic option for the treatment of the COVID-19 infection. Such an inhibitory strategy is currently successful in treating COVID-19 under FDA's emergency use authorization, although with limited benefit to the immunocompromised along with an unfortunate number of side effects and drug-drug interactions. Current COVID vaccines protect against severe disease and death but are mostly ineffective toward long COVID which has been seen in 5-36% of patients. SARS-CoV-2 is a rapidly mutating virus and is here to stay endemically. Hence, alternate therapeutics to treat SARS-CoV-2 infections are still needed. Moreover, because of the high degree of conservation of Mpro among different coronaviruses, any newly developed antiviral agents should better prepare us for potential future epidemics or pandemics. In this paper, we first describe the design and computational docking of a library of novel 188 first-generation peptidomimetic protease inhibitors using various electrophilic warheads with aza-peptide epoxides, α-ketoesters, and ß-diketones identified as the most effective. Second-generation designs, 192 compounds in total, focused on aza-peptide epoxides with drug-like properties, incorporating dipeptidyl backbones and heterocyclic ring motifs such as proline, indole, and pyrrole groups, yielding 8 hit candidates. These novel and specific inhibitors for SARS-CoV-2 Mpro can ultimately serve as valuable alternate and broad-spectrum antivirals against COVID-19.Communicated by Ramaswamy H. Sarma.

Human CYP2C19 Substrate and Inhibitor Characterization of Organophosphate Pesticides.

CYP2C19 is an important enzyme for organophosphate pesticide (OPP) metabolism. Because the OPPs can be both substrates and inhibitors of CYP2C19, we screened 45 OPPs for their ability to inhibit the activity of this enzyme and investigated the role of CYP2C19 in the metabolism of 22 of these molecules. We identified several nanomolar inhibitors of CYP2C19 as well as determined that thions, in general, are more potent inhibitors than oxons. We also determined that thions are readily metabolized by CYP2C19, although we saw no relationship between IC50 values and intrinsic clearance rates. This study may have implications for mitigating the risk of OPP poisoning.

Targeted Metabolomics of Organophosphate Pesticides and Chemical Warfare Nerve Agent Simulants Using High- and Low-Dose Exposure in Human Liver Microsomes.

Our current understanding of organophosphorus agent (pesticides and chemical warfare nerve agents) metabolism in humans is limited to the general transformation by cytochrome P450 enzymes and, to some extent, by esterases and paraoxonases. The role of compound concentrations on the rate of clearance is not well established and is further explored in the current study. We discuss the metabolism of 56 diverse organophosphorus compounds (both pesticides and chemical warfare nerve agent simulants), many of which were explored at two variable dose regimens (high and low), determining their clearance rates (Clint) in human liver microsomes. For compounds that were soluble at high concentrations, 1D-NMR, 31P, and MRM LC-MS/MS were used to calculate the Clint and the identity of certain metabolites. The determined Clint rates ranged from 0.001 to 2245.52 µL/min/mg of protein in the lower dose regimen and from 0.002 to 98.57 µL/min/mg of protein in the high dose regimen. Though direct equivalency between the two regimens was absent, we observed (1) both mono- and bi-phasic metabolism of the OPs and simulants in the microsomes. Compounds such as aspon and formothion exhibited biphasic decay at both high and low doses, suggesting either the involvement of multiple enzymes with different KM or substrate/metabolite effects on the metabolism. (2) A second observation was that while some compounds, such as dibrom and merphos, demonstrated a biphasic decay curve at the lower concentrations, they exhibited only monophasic metabolism at the higher concentration, likely indicative of saturation of some metabolic enzymes. (3) Isomeric differences in metabolism (between Z- and E- isomers) were also observed. (4) Lastly, structural comparisons using examples of the oxon group over the original phosphorothioate OP are also discussed, along with the identification of some metabolites. This study provides initial data for the development of in silico metabolism models for OPs with broad applications.

Fasting and diurnal blood ketonemia and glycemia responses to a six-week, energy-controlled ketogenic diet, supplemented with racemic R/S-BHB salts.

BACKGROUND: Single doses of exogenous ketone salts (KS) transiently increase circulating beta-hydroxybutyrate (BHB) (∼1 mM; 1-2 h) regardless of starting levels of ketosis; however, no studies have explored how sustained use of KS influences measures of ketonemia and glycemia. OBJECTIVES: To determine the response to a hypocaloric, well-formulated ketogenic diet (KD), with and without the inclusion of two daily racemic KS doses (6 g R-BHB + 6 g S-BHB per serving) on 1) daily fasting capillary R-BHB and glucose (R-BHB/GLUfast), 2) bi-weekly 13 h diurnal BHB and glucose (R-BHB/GLUdiur), 3) three-hours post-KS ingestion kinetics (R-BHBKS), and 4) bi-weekly fasting plasma enantiomer-specific BHB (R/S-BHBplasma). METHODS: Non-diabetic adults with overweight and obesity were randomized to receive a precisely measured hypocaloric KD (∼75 %en of maintenance) for six weeks, supplemented twice-daily with KS or placebo (PL). A non-randomized comparison group was provided an isonitrogenous/isoenergetic low-fat diet (LFD). All meals were provided to subjects. Capillary blood was collected daily to measure R-BHB/GLUfast and hourly for R-BHB/GLUdiur. Plasma was collected to measure R/S-BHBplasma, insulin, fasting glucose, and insulin resistance (HOMA-IR). Total AUC was calculated using the trapezoidal method. RESULTS: Mean R-BHBfast increased significantly during KD + PL (1.0 mM BHB), an effect enhanced 26% during KD + KS. GLUfast AUC was -6% lower during KD + KS versus LFD. Mean R-BHBdiur increased 40% in KD + KS versus KD + PL, whereas GLUdiur decreased 13% during both KDs versus LFD. R-BHBKS peaked (Δ: ∼1 mM) 1 h after the morning KS dose, but not following the afternoon dose. Both R/S-BHBplasma increased during KD independent of KS inclusion. R-BHBplasma was 50-times greater compared to S-BHBplasma, and the KS augmented S-BHBplasma 50% more than PL. Fasting insulin and HOMA-IR decreased after 14 days independent of diet. CONCLUSIONS: A hypocaloric KD was effective at reducing diurnal glucose compared to a LFD independent of weight loss, but twice-daily racemic KS ingestion during KD augmented ketonemia, both as R- and S-BHB, and decreased mean fasting glucose beyond a KD alone. The hypoglycemic effects of KD in combination with exogenous ketones merit further investigation.

Triple-combination therapy for cutaneous leishmaniasis using detergent-free, hyaluronate-coated elastic nanovesicles.

Aim: To develop and evaluate detergent-free, triple-drug-loaded, hyaluronate-coated elastic nanovesicles (H-ENVs) for the topical treatment of cutaneous leishmaniasis. Materials & methods: H-ENVs were developed and evaluated for vesicle size, entrapment efficiency, skin permeation and antileishmanial potential. Results: A 15.7 and 28.6% decrease in the cytotoxicity of paromomycin and amphotericin B, respectively, was observed in detergent-free ENVs compared with conventional ENVs. H-ENVs improved the efficacy of paromomycin against promastigote and amastigote models of leishmaniasis by 4- and 7.5-fold, respectively. In vivo investigation of H-ENVs demonstrated efficient topical management of cutaneous leishmaniasis. Conclusion: The results indicate the potential of H-ENVs as a safe topical treatment choice for cutaneous leishmaniasis.

Application of topical gel is an attractive alternative to oral or intravenous administration of drugs and is likely to deliver a higher dose of the drug to the target site with only rare systemic adverse effects. Nanotechnology-based topical drug delivery is an attractive aspect of pharmaceutical sciences that expresses interest in the topical treatment of cutaneous leishmaniasis. The authors' research focuses on the development and evaluation of novel multidrug-loaded, detergent-free nanovesicles for the simple and effective topical treatment of cutaneous leishmaniasis.

Influence of Nutritional Ketosis Achieved through Various Methods on Plasma Concentrations of Brain Derived Neurotropic Factor.

Brain-Derived Neurotropic Factor (BDNF) expression is decreased in conditions associated with cognitive decline as well as metabolic diseases. One potential strategy to improve metabolic health and elevate BDNF is by increasing circulating ketones. Beta-Hydroxybutyrate (BHB) stimulates BDNF expression, but the association of circulating BHB and plasma BDNF in humans has not been widely studied. Here, we present results from three studies that evaluated how various methods of inducing ketosis influenced plasma BDNF in humans. Study 1 determined BDNF responses to a single bout of high-intensity cycling after ingestion of a dose of ketone salts in a group of healthy adults who were habitually consuming either a mixed diet or a ketogenic diet. Study 2 compared how a ketogenic diet versus a mixed diet impacts BDNF levels during a 12-week resistance training program in healthy adults. Study 3 examined the effects of a controlled hypocaloric ketogenic diet, with and without daily use of a ketone-salt, on BDNF levels in overweight/obese adults. We found that (1) fasting plasma BDNF concentrations were lower in keto-adapted versus non keto-adapted individuals, (2) intense cycling exercise was a strong stimulus to rapidly increase plasma BDNF independent of ketosis, and (3) clinically significant weight loss was a strong stimulus to decrease fasting plasma BDNF independent of diet composition or level of ketosis. These results highlight the plasticity of plasma BDNF in response to lifestyle factors but does not support a strong association with temporally matched BHB concentrations.

Harnessing cortical plasticity via gabapentinoid administration promotes recovery after stroke.

Stroke causes devastating sensory-motor deficits and long-term disability due to disruption of descending motor pathways. Restoration of these functions enables independent living and therefore represents a high priority for those afflicted by stroke. Here, we report that daily administration of gabapentin, a clinically approved drug already used to treat various neurological disorders, promotes structural and functional plasticity of the corticospinal pathway after photothrombotic cortical stroke in adult mice. We found that gabapentin administration had no effects on vascular occlusion, haemodynamic changes nor survival of corticospinal neurons within the ipsilateral sensory-motor cortex in the acute stages of stroke. Instead, using a combination of tract tracing, electrical stimulation and functional connectivity mapping, we demonstrated that corticospinal axons originating from the contralateral side of the brain in mice administered gabapentin extend numerous collaterals, form new synaptic contacts and better integrate within spinal circuits that control forelimb muscles. Not only does gabapentin daily administration promote neuroplasticity, but it also dampens maladaptive plasticity by reducing the excitability of spinal motor circuitry. In turn, mice administered gabapentin starting 1 h or 1 day after stroke recovered skilled upper extremity function. Functional recovery persists even after stopping the treatment at 6 weeks following a stroke. Finally, chemogenetic silencing of cortical projections originating from the contralateral side of the brain transiently abrogated recovery in mice administered gabapentin, further supporting the conclusion that gabapentin-dependent reorganization of spared cortical pathways drives functional recovery after stroke. These observations highlight the strong potential for repurposing gabapentinoids as a promising treatment strategy for stroke repair.

Modeling neurodegenerative diseases with cerebral organoids and other three-dimensional culture systems: focus on Alzheimer's disease.

Many neurodegenerative diseases (NDs) such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis and Huntington's disease, are characterized by the progressive accumulation of abnormal proteinaceous assemblies in specific cell types and regions of the brain, leading to cellular dysfunction and brain damage. Although animal- and in vitro-based studies of NDs have provided the field with an extensive understanding of some of the mechanisms underlying these diseases, findings from these studies have not yielded substantial progress in identifying treatment options for patient populations. This necessitates the development of complementary model systems that are better suited to recapitulate human-specific features of ND pathogenesis. Three-dimensional (3D) culture systems, such as cerebral organoids generated from human induced pluripotent stem cells, hold significant potential to model NDs in a complex, tissue-like environment. In this review, we discuss the advantages of 3D culture systems and 3D modeling of NDs, especially AD and FTD. We also provide an overview of the challenges and limitations of the current 3D culture systems. Finally, we propose a few potential future directions in applying state-of-the-art technologies in 3D culture systems to understand the mechanisms of NDs and to accelerate drug discovery. Graphical abstract.

Optimization of TopoIV Potency, ADMET Properties, and hERG Inhibition of 5-Amino-1,3-dioxane-Linked Novel Bacterial Topoisomerase Inhibitors: Identification of a Lead with In Vivo Efficacy against MRSA.

Novel bacterial topoisomerase inhibitors (NBTIs) are among the most promising new antibiotics in preclinical/clinical development. We previously reported dioxane-linked NBTIs with potent antistaphylococcal activity and reduced hERG inhibition, a key safety liability. Herein, polarity-focused optimization enabled the delineation of clear structure-property relationships for both microsomal metabolic stability and hERG inhibition, resulting in the identification of lead compound 79. This molecule demonstrates potent antibacterial activity against diverse Gram-positive pathogens, inhibition of both DNA gyrase and topoisomerase IV, a low frequency of resistance, a favorable in vitro cardiovascular safety profile, and in vivo efficacy in a murine model of methicillin-resistant Staphylococcus aureus infection.

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.

Electrophysiological Maturation of Cerebral Organoids Correlates with Dynamic Morphological and Cellular Development.

Cerebral organoids (COs) are rapidly accelerating the rate of translational neuroscience based on their potential to model complex features of the developing human brain. Several studies have examined the electrophysiological and neural network features of COs; however, no study has comprehensively investigated the developmental trajectory of electrophysiological properties in whole-brain COs and correlated these properties with developmentally linked morphological and cellular features. Here, we profiled the neuroelectrical activities of COs over the span of 5 months with a multi-electrode array platform and observed the emergence and maturation of several electrophysiologic properties, including rapid firing rates and network bursting events. To complement these analyses, we characterized the complex molecular and cellular development that gives rise to these mature neuroelectrical properties with immunohistochemical and single-cell transcriptomic analyses. This integrated approach highlights the value of COs as an emerging model system of human brain development and neurological disease.

Design, synthesis, and in vitro evaluation of aza-peptide aldehydes and ketones as novel and selective protease inhibitors.

Aza-peptide aldehydes and ketones are a new class of reversible protease inhibitors that are specific for the proteasome and clan CD cysteine proteases. We designed and synthesised aza-Leu derivatives that were specific for the chymotrypsin-like active site of the proteasome, aza-Asp derivatives that were effective inhibitors of caspases-3 and -6, and aza-Asn derivatives that inhibited S. mansoni and I. ricinus legumains. The crystal structure of caspase-3 in complex with our caspase-specific aza-peptide methyl ketone inhibitor with an aza-Asp residue at P1 revealed a covalent linkage between the inhibitor carbonyl carbon and the active site cysteinyl sulphur. Aza-peptide aldehydes and ketones showed no cross-reactivity towards cathepsin B or chymotrypsin. The initial in vitro selectivity of these inhibitors makes them suitable candidates for further development into therapeutic agents to potentially treat multiple myeloma, neurodegenerative diseases, and parasitic infections.

A Pre-Workout Supplement of Ketone Salts, Caffeine, and Amino Acids Improves High-Intensity Exercise Performance in Keto-Naïve and Keto-Adapted Individuals.

Background: Acute ingestion of ketone supplements alters metabolism and potentially exercise performance. No studies to date have evaluated the impact of co-ingestion of ketone salts with caffeine and amino acids on high intensity exercise performance, and no data exists in Keto-Adapted individuals.Methods: We tested the performance and metabolic effects of a pre-workout supplement containing beta-hydroxybutyrate (BHB) salts, caffeine, and amino acids (KCA) in recreationally-active adults habitually consuming a mixed diet (Keto-Naïve; n = 12) or a ketogenic diet (Keto-Adapted; n = 12). In a randomized and balanced manner, subjects consumed either the KCA consisting of ∼7 g BHB (72% R-BHB and 28% S-BHB) with ∼100 mg of caffeine, and amino acids (leucine and taurine) or Water (control condition) 15 minutes prior to performing a staged cycle ergometer time to exhaustion test followed immediately by a 30 second Wingate test.Results: Circulating total BHB concentrations increased rapidly after KCA ingestion in KN (154 to 732 µM) and KA (848 to 1,973 µM) subjects and stayed elevated throughout recovery in both groups. Plasma S-BHB increased >20-fold 15 minutes after KCA ingestion in both groups and remained elevated throughout recovery. Compared to Water, KCA ingestion increased time to exhaustion 8.3% in Keto-Naïve and 9.8% in Keto-Adapted subjects (P < 0.001). There was no difference in power output during the Wingate test between trials. Peak lactate immediately after exercise was higher after KCA (∼14.9 vs 12.7 mM).Conclusion: These results indicate that pre-exercise ingestion of a moderate dose of R- and S-BHB salts combined with caffeine, leucine and taurine improves high-intensity exercise performance to a similar extent in both Keto-Adapted and Keto-Naïve individuals.

Development and evaluation of novel miltefosine-polyphenol co-loaded second generation nano-transfersomes for the topical treatment of cutaneous leishmaniasis.

Objective: To test the hypothesis that miltefosine (MTF)-polyphenol co-loaded second-generation nano-transfersomes (SGNTs) can be an effective approach for the topical treatment of cutaneous leishmaniasis (CL).Methods: The co-loaded SGNTs with various MTF-polyphenol combinations were developed, evaluated and compared for the entrapment efficiency, vesicle size, deformability index, ex-vivo permeation, cytotoxicity, and anti-leishmanial potential, using both in-vitro and in-vivo models.Results: The co-loaded SGNTs were spherical in shape, with an average size of 119 ± 1.5 nm and a high entrapment efficiency of 73.7 ± 3.7%. The ex-vivo study displayed a 3.2-fold higher permeation of MTF when entrapped in co-loaded SGNTs, whereas cytotoxicity potential of co-loaded SGNTs was 43.2% higher than the MTF solution. A synergistic interaction was observed between MTF and apigenin (APG) among all polyphenols and an 8.0-fold lower IC50 was found against amastigotes of DsRed Leishmania mexicana, compared with the plain MTF solution. Moreover, the in-vivo studies displayed a 9.5-fold reduced parasitic burden in the L. mexicana infected BALB/c mice treated with MTF-APG co-loaded SGNTs gel.Conclusions: The potential of MTF-APG co-loaded SGNTs topical formulation is established for the first time as an effective drug delivery strategy against CL.

Topical treatment of cutaneous leishmaniasis with novel amphotericin B-miltefosine co-incorporated second generation ultra-deformable liposomes.

The present study aims to optimize and evaluate amphotericin B (AmB) and miltefosine (MTF) co-loaded second generation ultra-deformable liposomes (SGUDLs) for the topical treatment of cutaneous leishmaniasis (CL). The development of an effective topical drug formulation against CL is desirable because of its non-invasive nature, which may potentially enhance the patient adherence and treatment accessibility. AmB-MTF co-loaded SGUDLs were prepared and characterized for size, entrapment efficiency (EE) and elasticity. The optimized formulation was then subjected to ex-vivo permeation studies in addition to cytotoxicity and anti-leishmanial assays. The co-loaded SGUDLs had an average size of 139.7 ± 1.7 nm and high EE of 77.8 ± 3.9% with respect to AmB. The ex-vivo permeation of co-loaded SGUDLs exhibited 6.15-fold higher permeation of AmB. A synergistic interaction was observed between AmB and MTF, and anti-leishmanial activity of co-loaded SGUDLs against amastigotes of Lesihmania mexicana indicated 8.62 and 6.12-fold lower IC50 values of AmB and MTF as compared to plain drug solutions, respectively. The results of the in-vivo study displayed a significant reduction in the parasitic burden in an infected BALB/c experimental model of CL. In conclusion, AmB-MTF co-loaded SGUDLs could be an effective topical treatment option against CL.

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.

A Novel, Modified Human Butyrylcholinesterase Catalytically Degrades the Chemical Warfare Nerve Agent, Sarin.

Chemical warfare nerve agents (CWNAs) present a global threat to both military and civilian populations. The acute toxicity of CWNAs stems from their ability to effectively inhibit acetylcholinesterase (AChE). This inhibition can lead to uncontrolled cholinergic cellular signaling, resulting in cholinergic crisis and, ultimately, death. Although the current FDA-approved standard of care is moderately effective when administered early, development of novel treatment strategies is necessary. Butyrylcholinesterase (BChE) is an enzyme which displays a high degree of structural homology to AChE. Unlike AChE, the roles of BChE are uncertain and possibilities are still being explored. However, BChE appears to primarily serve as a bioscavenger of toxic esters due to its ability to accommodate a wide variety of substrates within its active site. Like AChE, BChE is also readily inhibited by CWNAs. Due to its high affinity for binding CWNAs, and that null-BChE yields no apparent health effects, exogenous BChE has been explored as a candidate therapeutic for CWNA intoxication. Despite years of research, minimal strides have been made to develop a catalytic bioscavenger. Furthermore, BChE is only in early clinical trials as a stoichiometric bioscavenger of CWNAs, and large quantities must be administered to treat CWNA toxicity. Here, we describe previously unidentified mutations to residues within and adjacent to the acyl binding pocket (positions 282-285 were mutagenized from YGTP to NHML) of BChE that confer catalytic degradation of the CWNA, sarin. These mutations, along with corresponding future efforts, may finally lead to a novel therapeutic to combat CWNA intoxication.