In Vitro and In Vivo Characterization of Tebipenem (TBP), an Orally Active Carbapenem, against Biothreat Pathogens.

Bacillus anthracis and Yersinia pestis, causative pathogens for anthrax and plague, respectively, along with Burkholderia mallei and B. pseudomallei are potential bioterrorism threats. Tebipenem pivoxil hydrobromide (TBP HBr, formerly SPR994), is an orally available prodrug of tebipenem, a carbapenem with activity versus multidrug-resistant (MDR) gram-negative pathogens, including quinolone-resistant and extended-spectrum-ß-lactamase-producing Enterobacterales. We evaluated the in vitro activity and in vivo efficacy of tebipenem against biothreat pathogens. Tebipenem was active in vitro against 30-strain diversity sets of B. anthracis, Y. pestis, B. mallei, and B. pseudomallei with minimum inhibitory concentration (MIC) values of 0.001 - 0.008 µg/ml for B. anthracis, ≤0.0005 - 0.03 µg/ml for Y. pestis, 0.25 - 1 µg/ml for B. mallei, and 1 - 4 µg/ml for B. pseudomallei In a B. anthracis murine model, all control animals died within 52 h post challenge. The survival rates in the groups treated with tebipenem were 75% and 73% when dosed at 12 h and 24 h post challenge, respectively. The survival rates in the positive control groups treated with ciprofloxacin were 75% and when dosed 12 h and 25% when dosed 24 h post challenge, respectively. Survival rates were significantly (p=0.0009) greater in tebipenem groups treated at 12 h and 24 h post challenge and in the ciprofloxacin group 12 h post-challenge vs. the vehicle-control group. For Y. pestis, survival rates for all animals in the tebipenem and ciprofloxacin groups were significantly (p<0.0001) greater than the vehicle-control group. These results support further development of tebipenem for treating biothreat pathogens.

Early detection of Ebola virus proteins in peripheral blood mononuclear cells from infected mice.

BACKGROUND: Detection of viral ribo-nucleic acid (RNA) via real-time polymerase chain reaction (RT-PCR) is the gold standard for the detection of Ebola virus (EBOV) during acute infection. However, the earliest window for viral RNA detection in blood samples is 48-72 h post-onset of symptoms. Therefore, efforts to develop additional orthogonal assays using complementary immunological and serological technologies are still needed to provide simplified methodology for field diagnostics. Furthermore, unlike RT-PCR tests, immunoassays that target viral proteins and/or early host responses are less susceptible to sequence erosion due to viral genetic drift. Although virus is shed into the bloodstream from infected cells, the wide dynamic range of proteins in blood plasma makes this a difficult sample matrix for the detection of low-abundant viral proteins. We hypothesized that the isolation of peripheral blood mononuclear cells (PBMCs), which are the first cellular targets of the Ebola virus (EBOV), may provide an enriched source of viral proteins. METHODS: A mouse infection model that employs a mouse-adapted EBOV (MaEBOV) was chosen as a proof-of-principal experimental paradigm to determine if viral proteins present in PBMCs can help diagnose EBOV infection pre-symptomatically. We employed a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) platform to provide both high sensitivity and specificity for the detection and relative quantitation of viral proteins in PBMCs collected during MaEBOV infection. Blood samples pooled from animals at the post-infection time-points were used to determine the viral load by RT-PCR and purify PBMCs. RESULTS: Using quantitative LC-MS/MS, we detected two EBOV proteins (vp40 and nucleoprotein) in samples collected on Day 2 post-infection, which was also the first day of detectable viremia via RT-PCR. These results were confirmed via western blot which was performed on identical PBMC lysates from each post-infection time point. CONCLUSIONS: While mass spectrometry is not currently amenable to field diagnostics, these results suggest that viral protein enrichment in PBMCs in tandem with highly sensitive immunoassays platforms, could lead to the development of a rapid, high-throughput diagnostic platform for pre-symptomatic detection of EBOV infection.

Countering Zika Virus: The USAMRIID Response.

The United States Army Medical Research Institute of Infectious Diseases (USAMRIID) possesses an array of expertise in diverse capabilities for the characterization of emerging infectious diseases from the pathogen itself to human or animal infection models. The recent Zika virus (ZIKV) outbreak was a challenge and an opportunity to put these capabilities to work as a cohesive unit to quickly respond to a rapidly developing threat. Next-generation sequencing was used to characterize virus stocks and to understand the introduction and spread of ZIKV in the United States. High Content Imaging was used to establish a High Content Screening process to evaluate antiviral therapies. Functional genomics was used to identify critical host factors for ZIKV infection. An animal model using the temporal blockade of IFN-I in immunocompetent laboratory mice was investigated in conjunction with Positron Emission Tomography to study ZIKV. Correlative light and electron microscopy was used to examine ZIKV interaction with host cells in culture and infected animals. A quantitative mass spectrometry approach was used to examine the protein and metabolite type or concentration changes that occur during ZIKV infection in blood, cells, and tissues. Multiplex fluorescence in situ hybridization was used to confirm ZIKV replication in mouse and NHP tissues. The integrated rapid response approach developed at USAMRIID presented in this review was successfully applied and provides a new template pathway to follow if a new biological threat emerges. This streamlined approach will increase the likelihood that novel medical countermeasures could be rapidly developed, evaluated, and translated into the clinic.

Clinical Laboratory Values as Early Indicators of Ebola Virus Infection in Nonhuman Primates.

The Ebola virus (EBOV) outbreak in West Africa during 2013-2016 demonstrated the need to improve Ebola virus disease (EVD) diagnostics and standards of care. This retrospective study compared laboratory values and clinical features of 3 nonhuman primate models of lethal EVD to assess associations with improved survival time. In addition, the study identified laboratory values useful as predictors of survival, surrogates for EBOV viral loads, and triggers for initiation of therapeutic interventions in these nonhuman primate models. Furthermore, the data support that, in nonhuman primates, the Makona strain of EBOV may be less virulent than the Kikwit strain of EBOV. The applicability of these findings as potential diagnostic and management tools for EVD in humans warrants further investigation.

Efficacy and safety of mavrilimumab in subjects with rheumatoid arthritis.

OBJECTIVES: Mavrilimumab, a human monoclonal antibody targeting the alpha subunit of the granulocyte-macrophage colony-stimulating factor receptor, was evaluated in a phase 2 randomised, double-blind, placebo-controlled study to investigate efficacy and safety in subjects with rheumatoid arthritis (RA). METHODS: Subcutaneous mavrilimumab (10 mg, 30 mg, 50 mg, or 100 mg) or placebo was administered every other week for 12 weeks in subjects on stable background methotrexate therapy. The primary endpoint was the proportion of subjects achieving a ≥1.2 decrease from baseline in Disease Activity Score (DAS28-CRP) at week 12. RESULTS: 55.7% of mavrilimumab-treated subjects met the primary endpoint versus 34.7% placebo (p=0.003) at week 12; for the 10 mg, 30 mg, 50 mg, and 100 mg groups, responses were 41.0% (p=0.543), 61.0% (p=0.011), 53.8% (p=0.071), and 66.7% (p=0.001) respectively. Response rate differences from placebo were observed at week 2 and increased throughout the treatment period. The 100 mg dose demonstrated a significant effect versus placebo on DAS28-CRP<2.6 (23.1% vs 6.7%, p=0.016), all categories of the American College of Rheumatology (ACR) criteria (ACR20: 69.2% vs 40.0%, p=0.005; ACR50: 30.8% vs 12.0%, p=0.021; ACR70: 17.9% vs 4.0%, p=0.030), and the Health Assessment Questionnaire Disability Index (-0.48 vs -0.25, p=0.005). A biomarker-based disease activity score showed a dose-dependent decrease at week 12, indicating suppression of disease-related biological pathways. Adverse events were generally mild or moderate in intensity. No significant hypersensitivity reactions, serious or opportunistic infections, or changes in pulmonary parameters were observed. CONCLUSIONS: Mavrilimumab induced rapid clinically significant responses in RA subjects, suggesting that inhibiting the mononuclear phagocyte pathway may provide a novel therapeutic approach for RA.

The development of broad-spectrum antiviral medical countermeasures to treat viral hemorrhagic fevers caused by natural or weaponized virus infections.

The Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense (JPEO-CBRND) began development of a broad-spectrum antiviral countermeasure against deliberate use of high-consequence viral hemorrhagic fevers (VHFs) in 2016. The effort featured comprehensive preclinical research, including laboratory testing and rapid advancement of lead molecules into nonhuman primate (NHP) models of Ebola virus disease (EVD). Remdesivir (GS-5734, Veklury, Gilead Sciences) was the first small molecule therapeutic to successfully emerge from this effort. Remdesivir is an inhibitor of RNA-dependent RNA polymerase, a viral enzyme that is essential for viral replication. Its robust potency and broad-spectrum antiviral activity against certain RNA viruses including Ebola virus and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) led to its clinical evaluation in randomized, controlled trials (RCTs) in human patients during the 2018 EVD outbreak in the Democratic Republic of the Congo (DRC) and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic today. Remdesivir was recently approved by the US Food and Drug Administration (FDA) for the treatment of COVID-19 requiring hospitalization. Substantial gaps remain in improving the outcomes of acute viral infections for patients afflicted with both EVD and COVID-19, including how to increase therapeutic breadth and strategies for the prevention and treatment of severe disease. Combination therapy that joins therapeutics with complimentary mechanisms of action appear promising, both preclinically and in RCTs. Importantly, significant programmatic challenges endure pertaining to a clear drug and biological product development pathway for therapeutics targeting biodefense and emerging pathogens when human efficacy studies are not ethical or feasible. For example, remdesivir's clinical development was facilitated by outbreaks of Ebola and SARS-CoV-2; as such, the development pathway employed for remdesivir is likely to be the exception rather than the rule. The current regulatory licensure pathway for therapeutics targeting rare, weaponizable VHF agents is likely to require use of FDA's established Animal Rule (21 CFR 314.600-650 for drugs; 21 CFR 601.90-95 for biologics). The FDA may grant marketing approval based on adequate and well-controlled animal efficacy studies when the results of those studies establish that the drug is safe and likely to produce clinical benefit in humans. In practical terms, this is anticipated to include a series of rigorous, well-documented, animal challenge studies, to include aerosol challenge, combined with human safety data. While small clinical studies against naturally occurring, high-consequence pathogens are typically performed where possible, approval for the therapeutics currently under development against biodefense pathogens will likely require the Animal Rule pathway utilizing studies in NHPs. We review the development of remdesivir as illustrative of the effort that will be needed to field future therapeutics against highly lethal, infectious agents.

Remdesivir is efficacious in rhesus monkeys exposed to aerosolized Ebola virus.

Efficacious therapeutics for Ebola virus disease are in great demand. Ebola virus infections mediated by mucosal exposure, and aerosolization in particular, present a novel challenge due to nontypical massive early infection of respiratory lymphoid tissues. We performed a randomized and blinded study to compare outcomes from vehicle-treated and remdesivir-treated rhesus monkeys in a lethal model of infection resulting from aerosolized Ebola virus exposure. Remdesivir treatment initiated 4 days after exposure was associated with a significant survival benefit, significant reduction in serum viral titer, and improvements in clinical pathology biomarker levels and lung histology compared to vehicle treatment. These observations indicate that remdesivir may have value in countering aerosol-induced Ebola virus disease.

Vinyl Sulfone-Based Inhibitors of Nonstructural Protein 2 Block the Replication of Venezuelan Equine Encephalitis Virus.

Emerging infectious diseases like those caused by arboviruses such as Venezuelan equine encephalitis virus (VEEV) pose a serious threat to public health systems. Development of medical countermeasures against emerging infectious diseases are of utmost importance. In this work, an acrylate and vinyl sulfone-based chemical series was investigated as promising starting scaffolds against VEEV and as inhibitors of the cysteine protease domain of VEEV's nonstructural protein 2 (nsP2). Primary screen and dose response studies were performed to evaluate the potency and cytotoxicity of the compounds. The results provide structural insights into a new class of potent nonpeptidic covalent inhibitors of nsP2 cysteine protease represented by compound 11 (VEEV TrD, EC50 = 2.4 µM (HeLa), 1.6 µM (Vero E6)). These results may facilitate the evolution of the compounds into selective and broad-spectrum anti-alphaviral drug leads.

Recent successes in therapeutics for Ebola virus disease: no time for complacency.

The PALM trial in the Democratic Republic of the Congo identified a statistically significant survival benefit for two monoclonal antibody-based therapeutics in the treatment of acute Ebola virus disease; however, substantial gaps remain in improving the outcomes of acute Ebola virus disease and for the survivors. Ongoing efforts are needed to develop more effective strategies, particularly for individuals with severe disease, for prevention and treatment of viral persistence in immune-privileged sites, for optimisation of post-exposure prophylaxis, and to increase therapeutic breadth. As antibody-based approaches are identified and advanced, promising small-molecule antivirals currently in clinical stage development should continue to be evaluated for filovirus diseases, with consideration of their added value in combination approaches with bundled supportive care, their penetration in tissues of interest, the absence of interaction with glycoprotein-based vaccines, and filoviral breadth.

Molecular characterization of novel and selective peroxisome proliferator-activated receptor alpha agonists with robust hypolipidemic activity in vivo.

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) is recognized as the primary target of the fibrate class of hypolipidemic drugs and mediates lipid lowering in part by activating a transcriptional cascade that induces genes involved in the catabolism of lipids. We report here the characterization of three novel PPARalpha agonists with therapeutic potential for treating dyslipidemia. These structurally related compounds display potent and selective binding to human PPARalpha and support robust recruitment of coactivator peptides in vitro. These compounds markedly potentiate chimeric transcription systems in cell-based assays and strikingly lower serum triglycerides in vivo. The transcription networks induced by these selective PPARalpha agonists were assessed by transcriptional profiling of mouse liver after short- and long-term treatment. The induction of several known PPARalpha target genes involved with fatty acid metabolism were observed, reflecting the expected pharmacology associated with PPARalpha activation. We also noted the down-regulation of a number of genes related to immune cell function, the acute phase response, and glucose metabolism, suggesting that these compounds may have anti-inflammatory action in the mammalian liver. Whereas these compounds are efficacious in acute preclinical models, extended safety studies and further clinical testing will be required before the full therapeutic promise of a selective PPARalpha agonist is realized.

Synthesis and evaluation of novel alpha-heteroaryl-phenylpropanoic acid derivatives as PPARalpha/gamma dual agonists.

The synthesis of a new series of phenylpropanoic acid derivatives incorporating an heteroaryl group at the alpha-position and their evaluation for binding and activation of PPARalpha and PPARgamma are presented in this report. Among the new compounds, (S)-3-{4-[3-(5-methyl-2-phenyl-oxazol-4-yl)-propyl]-phenyl}-2-1,2,3-triazol-2-yl-propionic acid (17j), was identified as a potent human PPARalpha/gamma dual agonist (EC(50)=0.013 and 0.061 microM, respectively) with demonstrated oral bioavailability in rat and dog. 17j was shown to decrease insulin levels, plasma glucose, and triglycerides in the ZDF female rat model. In the human apolipoprotein A-1/CETP transgenic mouse model 17j produced increases in hApoA1 and HDL-C and decreases in plasma triglycerides. The increased potency for binding and activation of both PPAR subtypes observed with 17j when compared to previous analogs in this series was explained based on results derived from crystallographic and modeling studies.

Cholesterol-conjugated stapled peptides inhibit Ebola and Marburg viruses in vitro and in vivo.

Filoviridae currently includes five official and one proposed genera. Genus Ebolavirus includes five established and one proposed ebolavirus species for Bombali virus (BOMV), Bundibugyo virus (BDBV), Ebola virus (EBOV), Reston virus (RESTV), Sudan virus (SUDV) and Taï Forest virus (TAFV), and genus Marburgvirus includes a single species for Marburg virus (MARV) and Ravn virus (RAVV). Ebola virus (EBOV) has emerged as a significant public health concern since the 2013-2016 Ebola Virus Disease outbreak in Western Africa. Currently, there are no therapeutics approved and the need for Ebola-specific therapeutics remains a gap. In search for anti-Ebola therapies we tested the idea of using inhibitory properties of peptides corresponding to the C-terminal heptad-repeat (HR2) domains of class I fusion proteins against EBOV infection. The fusion protein GP2 of EBOV belongs to class I, suggesting that a similar strategy to HIV may be applied to inhibit EBOV infection. The serum half-life of peptides was expanded by cholesterol conjugation to allow daily dosing. The peptides were further constrained to stabilize a helical structure to increase the potency of inhibition. The EC50s of lead peptides were in low micromolar range, as determined by a high-content imaging test of EBOV-infected cells. Lead peptides were tested in an EBOV lethal mouse model and efficacy of the peptides were determined following twice-daily administration of peptides for 9 days. The most potent peptide was able to protect mice from lethal challenge of mouse-adapted Ebola virus. These data show that engineered peptides coupled with cholesterol can inhibit viral production, protect mice against lethal EBOV infection, and may be used to build novel therapeutics against EBOV.

Effects of modifications of the linker in a series of phenylpropanoic acid derivatives: Synthesis, evaluation as PPARalpha/gamma dual agonists, and X-ray crystallographic studies.

A new series of alpha-aryl or alpha-heteroarylphenyl propanoic acid derivatives was synthesized that incorporate acetylene-, ethylene-, propyl-, or nitrogen-derived linkers as a replacement of the commonly used ether moiety that joins the central phenyl ring with the lipophilic tail. The effect of these modifications in the binding and activation of PPARalpha and PPARgamma was first evaluated in vitro. Compounds possessing suitable profiles were then evaluated in the ob/ob mouse model of type 2 diabetes. The propylene derivative 40 and the propyl derivative 53 demonstrated robust plasma glucose lowering activity in this model. Compound 53 was also evaluated in male Zucker diabetic fatty rats and was found to achieve normalization of glucose, triglycerides, and insulin levels. An X-ray crystal structure of the complex of 53 with the PPARgamma-ligand-binding domain was obtained and discussed in this report.

African and Asian Zika Virus Isolates Display Phenotypic Differences Both In Vitro and In Vivo.

Zika virus (ZIKV) is a mosquito-borne member of the genus Flavivirus that has emerged since 2007 to cause outbreaks in Africa, Asia, Oceania, and most recently, in the Americas. Here, we used an isolate history as well as genetic and phylogenetic analyses to characterize three low-passage isolates representing African (ArD 41525) and Asian (CPC-0740, SV0127-14) lineages to investigate the potential phenotypic differences in vitro and in vivo. The African isolate displayed a large plaque phenotype (∼3-4 mm) on Vero and HEK-293 cells, whereas the Asian isolates either exhibited a small plaque phenotype (∼1-2 mm) or did not produce any plaques. In multistep replication kinetics in nine different vertebrate and insect cell lines, the African isolate consistently displayed faster replication kinetics and yielded ∼10- to 10,000-fold higher peak virus titers (infectious or RNA copies) compared with the Asian isolates. Oral exposure of Aedes aegypti mosquitoes with the African isolate yielded higher infection and dissemination rates compared with the Asian isolates. Infection of Ifnar1-/- mice with the African isolate produced a uniformly fatal disease, whereas infection with the Asian isolates produced either a delay in time-to-death or a significantly lower mortality rate. Last, the African isolate was > 10,000-fold more virulent than the Asian isolates in an interferon type I antibody blockade mouse model. These data demonstrate substantial phenotypic differences between low-passage African and Asian isolates both in vitro and in vivo and warrant further investigation. They also highlight the need for basic characterization of ZIKV isolates, as the utilization of the uncharacterized isolates could have consequences for animal model and therapeutic/vaccine development.

Differential regulation of the cynomolgus, human, and rat acyl-CoA oxidase promoters by PPARalpha.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear receptor family of transcription factors and is recognized as the molecular target of the hypolipidemic fibrate drugs. Fibrates promote lipid catabolism by inducing genes involved in fatty acid beta-oxidation. In rodents this is accompanied by peroxisome proliferation, and after chronic dosing hepatocarcinoma, whereas epidemiological studies suggest these adverse events are lacking in humans. Rodents such as rats and mice appear particularly sensitive to PPARalpha-induced peroxisome proliferation while humans are resistant. These findings question the utility of rodent models for safety monitoring of experimental PPARalpha agonists and highlight the need for additional preclinical models that display greater physiological relevance for human response. Thus we have focused on elucidating the molecular mechanism of species-dependent peroxisome proliferation by understanding the PPARalpha-dependent regulation of the acyl-CoA oxidase (AOX) promoter, the rate-limiting step of peroxisomal beta-oxidation. We have chosen the cynomolgus monkey as a model that is modestly responsive to peroxisome proliferation and functionally characterized it against the highly responsive rat and non-responsive human species. We report the identification of a putative peroxisome proliferator response element (PPRE) within the 2.3 kb proximal promoter of the cynomolgus monkey AOX gene. Characterization of these promoters using a series of constitutively active, PPARalpha constructs demonstrate that the PPREs within the proximal cynomolgus and human AOX promoters are non-responsive to PPARalpha whereas the rat PPRE is highly responsive. These findings were verified in vivo using a small molecule PPARalpha agonist. Taken together, we demonstrate concordant regulation of the AOX promoter by PPARalpha in cynomolgus monkeys and humans and suggest that this model is superior to rodent models with respect to preclinical evaluation of PPARalpha agonists.

Searching for Therapeutics Against Botulinum Neurotoxins: A True Challenge for Drug Discovery.

Botulinum neurotoxins (BoNTs), the most potent known toxins, cause severe muscle paralysis and death at nanogram exposures and are considered biothreat agents. BoNTs target the neuromuscular junction where they release smaller zinc metalloprotease light chains (LCs) into the neuron cytosol that selectively cleave SNARE proteins and thus block the exocytosis of acetylcholine neurotransmitters necessary for skeletal muscle contraction. The majority of efforts to develop post-symptomatic therapeutics for botulism poisoning have focused on inhibiting the LC and tremendous strides have been made in understanding how the LC binds to the SNARE proteins via X-ray crystallography. Subsequent homology modeling and structure based drug design have led to the discovery of multiple small molecule BoNT/A inhibitors in the 0.05 ~10 µΜ range, but to date none have shown significant post-symptomatic efficacy in an animal model of botulinum intoxication. With the lack of reported pharmacokinetic data, we have analyzed the BoNT/A inhibitor lead chemical matter from a physicochemical property point of view and have attempted to understand if bioavailability of drug at the neuromuscular junction is the root cause of this apparent in vitro/in vivo disconnect in the field.

Novel therapeutic uses and formulations of botulinum neurotoxins: a patent review (2012 - 2014).

INTRODUCTION: Botulinum neurotoxins (BoNTs) are among the most toxic of known biological molecules and function as acetylcholine release inhibitors and neuromuscular blocking agents. Paradoxically, these properties also make them valuable therapeutic agents for the treatment of movement disorders, urological conditions and hypersecretory disorders. Greater understanding of their molecular mechanism of action and advances in protein engineering has led to significant efforts to improve and expand their function with a view towards broadening their therapeutic potential. AREAS COVERED: Searches of Espacenet and Google Patent have revealed a number of patents related to BoNTs. This review will focus on novel therapeutic uses and formulations disclosed during 2012 - 2014. The seven patents discussed will include nanoformulations of FDA-approved BoNTs, additional BoNT subtypes and novel BoNT variants and chimeras created through protein engineering. Supporting patents and related publications are also briefly discussed. EXPERT OPINION: The clinical and commercial success of BoNTs has prompted investigation into novel BoNTs or BoNT-mediated chimeras with promising in vitro results. Distinct strategies including the use of nanoformulations and targeted delivery have been implemented to identify new indication and improved functionality. Greater understanding of their systemic exposure, efficacy and safety profiles will be required for further development.

Phosphatase Inhibitors Function as Novel, Broad Spectrum Botulinum Neurotoxin Antagonists in Mouse and Human Embryonic Stem Cell-Derived Motor Neuron-Based Assays.

There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT) poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC). Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s) of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs). Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.

SRC family kinase inhibitors antagonize the toxicity of multiple serotypes of botulinum neurotoxin in human embryonic stem cell-derived motor neurons.

Botulinum neurotoxins (BoNTs), the causative agents of botulism, are potent inhibitors of neurotransmitter release from motor neurons. There are currently no drugs to treat BoNT intoxication after the onset of the disease symptoms. In this study, we explored how modulation of key host pathways affects the process of BoNT intoxication in human motor neurons, focusing on Src family kinase (SFK) signaling. Motor neurons derived from human embryonic stem (hES) cells were treated with a panel of SFK inhibitors and intoxicated with BoNT serotypes A, B, or E (which are responsible for >95 % of human botulism cases). Subsequently, it was found that bosutinib, dasatinib, KX2-391, PP1, PP2, Src inhibitor-1, and SU6656 significantly antagonized all three of the serotypes. Furthermore, the data indicated that the treatment of hES-derived motor neurons with multiple SFK inhibitors increased the antagonistic effect synergistically. Mechanistically, the small molecules appear to inhibit BoNTs by targeting host pathways necessary for intoxication and not by directly inhibiting the toxins' proteolytic activity. Importantly, the identified inhibitors are all well-studied with some in clinical trials while others are FDA-approved drugs. Overall, this study emphasizes the importance of targeting host neuronal pathways, rather than the toxin's enzymatic components, to antagonize multiple BoNT serotypes in motor neurons.

Recent advances in botulinum neurotoxin inhibitor development.

Botulinum neurotoxins (BoNTs) are endopeptidases that target motor neurons and block acetylcholine neurotransmitter release. This action results in the muscle paralysis that defines the disease botulism. To date, there are no FDA-approved therapeutics to treat BoNT-mediated paralysis after intoxication of the motor neuron. Importantly, the rationale for pursuing treatments to counter these toxins is driven by their potential misuse. Current drug discovery efforts have mainly focused on small molecules, peptides, and peptidomimetics that can directly and competitively inhibit BoNT light chain proteolytic activity. Although this is a rational approach, direct inhibition of the Zn(2+) metalloprotease activity has been elusive as demonstrated by the dearth of candidates undergoing clinical evaluation. Therefore, broadening the scope of viable targets beyond that of active site protease inhibitors represents an additional strategy that could move the field closer to the clinic. Here we review the rationale, and discuss the outcomes of earlier approaches and highlight potential new targets for BoNT inhibition. These include BoNT uptake and processing inhibitors, enzymatic inhibitors, and modulators of neuronal processes associated with toxin clearance, neurotransmitter potentiation, and other pathways geared towards neuronal recovery and repair.