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1.
Antiviral Res ; 208: 105444, 2022 12.
Article in English | MEDLINE | ID: mdl-36243175

ABSTRACT

Infections by pathogenic New World mammarenaviruses (NWM)s, including Junín virus (JUNV), can result in a severe life-threatening viral hemorrhagic fever syndrome. In the absence of FDA-licensed vaccines or antivirals, these viruses are considered high priority pathogens. The mammarenavirus envelope glycoprotein complex (GPC) mediates pH-dependent fusion between viral and cellular membranes, which is essential to viral entry and may be vulnerable to small-molecule inhibitors that disrupt this process. ARN-75039 is a potent fusion inhibitor of a broad spectrum of pseudotyped and native mammarenaviruses in cell culture and Tacaribe virus infection in mice. In the present study, we evaluated ARN-75039 against pathogenic JUNV in the rigorous guinea pig infection model. The compound was well-tolerated and had favorable pharmacokinetics supporting once-per-day oral dosing in guinea pigs. Importantly, significant protection against JUNV challenge was observed even when ARN-75039 was withheld until 6 days after the viral challenge when clinical signs of disease are starting to develop. We also show that ARN-75039 combination treatment with favipiravir, a viral polymerase inhibitor, results in synergistic activity in vitro and improves survival outcomes in JUNV-challenged guinea pigs. Our findings support the continued development of ARN-75039 as an attractive therapeutic candidate for treating mammarenaviral hemorrhagic fevers, including those associated with NWM infection.


Subject(s)
Arenaviridae , Hemorrhagic Fever, American , Hemorrhagic Fevers, Viral , Junin virus , Guinea Pigs , Mice , Animals , Hemorrhagic Fever, American/drug therapy , Pyrazines/pharmacology , Pyrazines/therapeutic use , Amides/pharmacology , Amides/therapeutic use , Anti-Retroviral Agents/pharmacology
2.
Antiviral Res ; 193: 105125, 2021 09.
Article in English | MEDLINE | ID: mdl-34197863

ABSTRACT

Several arenaviruses, including Lassa and Lujo viruses in Africa and five New World arenavirus (NWA) species in the Americas, cause life-threatening viral hemorrhagic fevers. In the absence of licensed antiviral therapies, these viruses pose a significant public health risk. The envelope glycoprotein complex (GPC) mediates arenavirus entry through a pH-dependent fusion of the viral and host endosomal membranes. It thus is recognized as a viable target for small-molecule fusion inhibitors. Here, we report on the antiviral activity and pre-clinical development of the novel broad-spectrum arenavirus fusion inhibitors, ARN-75039 and ARN-75041. In Tacaribe virus (TCRV) pseudotyped and native virus assays, the ARN compounds were active in the low to sub-nanomolar range with selectivity indices exceeding 1000. Pharmacokinetic analysis of the orally administered compounds revealed an extended half-life in mice supporting once-daily dosing, and the compounds were well tolerated at the highest tested dose of 100 mg/kg. In a proof-of-concept prophylactic efficacy study, doses of 10 and 35 mg/kg of either compound dramatically improved survival outcome and potently inhibited TCRV replication in serum and various tissues. Additionally, in contrast to surviving mice that received ribavirin or placebo, animals treated with ARN-75039 or ARN-75041 were cured of TCRV infection. In a follow-up study with ARN-75039, impressive therapeutic efficacy was demonstrated under conditions where treatment was withheld until after the onset of disease. Taken together, the data strongly support the continued development of ARN-75039 as a candidate therapeutic for the treatment of severe arenaviral diseases.


Subject(s)
Antiviral Agents/pharmacology , Arenaviridae Infections/drug therapy , Arenaviruses, New World/drug effects , Membrane Fusion/drug effects , Small Molecule Libraries/pharmacology , Administration, Oral , Animals , Antiviral Agents/pharmacokinetics , Chlorocebus aethiops , Male , Mice , Ribavirin/pharmacology , Small Molecule Libraries/pharmacokinetics , Vero Cells , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism , Virus Internalization/drug effects
3.
Bioorg Med Chem Lett ; 41: 127983, 2021 06 01.
Article in English | MEDLINE | ID: mdl-33965007

ABSTRACT

We identified and explored the structure-activity relationship (SAR) of a novel heterocyclic chemical series of arenavirus cell entry inhibitors. Optimized lead compounds, including diphenyl-substituted imidazo[1,2-a]pyridines, benzimidazoles, and benzotriazoles exhibited low to sub-nanomolar potency against both pseudotyped and infectious Old and New World arenaviruses, attractive metabolic stability in human and most nonhuman liver microsomes as well as a lack of hERG K + channel or CYP enzyme inhibition. Moreover, the straightforward synthesis of several lead compounds (e.g., the simple high yield 3-step synthesis of imidazo[1,2-a]pyridine 37) could provide a cost-effective broad-spectrum arenavirus therapeutic that may help to minimize the cost-prohibitive burdens associated with treatments for emerging viruses in economically challenged geographical settings.


Subject(s)
Antiviral Agents/pharmacology , Arenavirus/drug effects , Drug Discovery , Heterocyclic Compounds/pharmacology , Viral Envelope Proteins/antagonists & inhibitors , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Arenavirus/metabolism , Dose-Response Relationship, Drug , Heterocyclic Compounds/chemical synthesis , Heterocyclic Compounds/chemistry , Humans , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity Relationship , Viral Envelope Proteins/metabolism
4.
ACS Med Chem Lett ; 11(6): 1160-1167, 2020 Jun 11.
Article in English | MEDLINE | ID: mdl-32550996

ABSTRACT

We identified and explored the structure-activity-relationship (SAR) of an adamantane carboxamide chemical series of Ebola virus (EBOV) inhibitors. Selected analogs exhibited half-maximal inhibitory concentrations (EC50 values) of ∼10-15 nM in vesicular stomatitis virus (VSV) pseudotyped EBOV (pEBOV) infectivity assays, low hundred nanomolar EC50 activity against wild type EBOV, aqueous solubility >20 mg/mL, and attractive metabolic stability in human and nonhuman liver microsomes. X-ray cocrystallographic characterizations of a lead compound with the EBOV glycoprotein (GP) established the EBOV GP as a target for direct compound inhibitory activity and further provided relevant structural models that may assist in identifying optimized therapeutic candidates.

5.
Bioorg Med Chem Lett ; 29(22): 126620, 2019 11 15.
Article in English | MEDLINE | ID: mdl-31537423

ABSTRACT

Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R1 and R4 scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R6 substituents and iterative R1, R4 and R6 substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.


Subject(s)
Antiviral Agents/pharmacology , Arenavirus/drug effects , Piperazines/pharmacology , Viral Envelope Proteins/antagonists & inhibitors , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Arenavirus/metabolism , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Molecular Structure , Piperazines/chemical synthesis , Piperazines/chemistry , Structure-Activity Relationship , Viral Envelope Proteins/metabolism
6.
Sci Rep ; 4: 5944, 2014 Aug 04.
Article in English | MEDLINE | ID: mdl-25089892

ABSTRACT

Influenza A viruses cause the respiratory illness influenza, which can be mild to fatal depending on the strain and host immune response. The flu polymerase acidic (PA), polymerase basic 1 (PB1), and polymerase basic 2 (PB2) proteins comprise the RNA-dependent RNA polymerase complex responsible for viral genome replication. The first crystal structures of the C-terminal domain of PA (PA-CTD) in the absence of PB1-derived peptides show a number of structural changes relative to the previously reported PB1-peptide bound structures. The human A/WSN/1933 (H1N1) and avian A/Anhui1/2013 (H7N9) strain PA-CTD proteins exhibit the same global topology as other strains in the absence of PB1, but differ extensively in the PB1 binding pocket including a widening of the binding groove and the unfolding of a ß-turn. Both PA-CTD proteins exhibited a significant increase in thermal stability in the presence of either a PB1-derived peptide or a previously reported inhibitor in differential scanning fluorimetry assays. These structural changes demonstrate plasticity in the PA-PB1 binding interface which may be exploited in the development of novel therapeutics.


Subject(s)
Influenza A Virus, H1N1 Subtype/chemistry , Influenza A Virus, H7N9 Subtype/chemistry , RNA-Dependent RNA Polymerase/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Influenza A Virus, H1N1 Subtype/enzymology , Influenza A Virus, H7N9 Subtype/enzymology , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication/physiology
7.
Proc Natl Acad Sci U S A ; 110(29): E2724-32, 2013 Jul 16.
Article in English | MEDLINE | ID: mdl-23818614

ABSTRACT

Voltage-gated sodium (Nav) channels play a fundamental role in the generation and propagation of electrical impulses in excitable cells. Here we describe two unique structurally related nanomolar potent small molecule Nav channel inhibitors that exhibit up to 1,000-fold selectivity for human Nav1.3/Nav1.1 (ICA-121431, IC50, 19 nM) or Nav1.7 (PF-04856264, IC50, 28 nM) vs. other TTX-sensitive or resistant (i.e., Nav1.5) sodium channels. Using both chimeras and single point mutations, we demonstrate that this unique class of sodium channel inhibitor interacts with the S1-S4 voltage sensor segment of homologous Domain 4. Amino acid residues in the "extracellular" facing regions of the S2 and S3 transmembrane segments of Nav1.3 and Nav1.7 seem to be major determinants of Nav subtype selectivity and to confer differences in species sensitivity to these inhibitors. The unique interaction region on the Domain 4 voltage sensor segment is distinct from the structural domains forming the channel pore, as well as previously characterized interaction sites for other small molecule inhibitors, including local anesthetics and TTX. However, this interaction region does include at least one amino acid residue [E1559 (Nav1.3)/D1586 (Nav1.7)] that is important for Site 3 α-scorpion and anemone polypeptide toxin modulators of Nav channel inactivation. The present study provides a potential framework for identifying subtype selective small molecule sodium channel inhibitors targeting interaction sites away from the pore region.


Subject(s)
Acetamides/pharmacology , Electrophysiological Phenomena/physiology , NAV1.3 Voltage-Gated Sodium Channel/metabolism , Thiazoles/pharmacology , Voltage-Gated Sodium Channel Blockers/pharmacology , Amino Acid Motifs/genetics , Binding Sites/genetics , HEK293 Cells , Humans , Inhibitory Concentration 50 , Molecular Sequence Data , NAV1.3 Voltage-Gated Sodium Channel/genetics , Patch-Clamp Techniques , Sequence Alignment
8.
Neurosci Lett ; 465(2): 138-42, 2009 Nov 13.
Article in English | MEDLINE | ID: mdl-19733209

ABSTRACT

The mammalian KCNQ (Kv7) gene family is composed of five members (KCNQ1-5). KCNQ2, Q4 and Q5 (KCNQ2-5) channels co-express with KCNQ3 to form heterotetrameric voltage-gated K(+) (KCNQ2-5/3) channels that underlie the endogenous M-current and regulate neuronal excitability in CNS and PNS neurons. Openers of one or a mixture of these channels may be an attractive therapeutic agent for epilepsy and pain. Non-selective KCNQ2-5/3 activators have shown efficacy in pre-clinical and clinical studies. However, more selective pharmacological profiles, including greater KCNQ sub-type-selective activation, could provide efficacy with fewer side effects. One such compound, ICA-27243, sub-type selectively enhances the activation of KCNQ2/3 channels and also exhibits efficacy in pre-clinical anticonvulsant models; Roeloffs et al. (2008) [15]; Wickenden et al. (2008) [27]. The binding site of non-selective KCNQ2-5/3 openers maps to the S5-S6 pore domain and is altered by mutation of a tryptophan residue (Trp236 in KCNQ2, Trp265 in KCNQ3) conserved among KCNQ2-5 channels; Schenzer et al. (2005) [19]; Wuttke et al. (2005) [30]. Here we report that the activity of the KCNQ2/3 selective opener ICA-27243 is not affected by these Trp mutations and does not map to the S5-S6 domain. Rather, the selective activity of ICA-27243 is determined by a novel site within the S1-S4 voltage-sensor domain (VSD) of KCNQ channels. The sub-type-selective activity of ICA-27243 may arise from greater sequence diversity of KCNQ family members within the ICA-27243 binding pocket, allowing for more selective small molecule-protein interactions.


Subject(s)
Benzamides/pharmacology , KCNQ2 Potassium Channel/metabolism , KCNQ3 Potassium Channel/metabolism , Membrane Transport Modulators/pharmacology , Pyridines/pharmacology , Amino Acid Sequence , Animals , Anticonvulsants/chemistry , Anticonvulsants/pharmacology , Benzamides/chemistry , Binding Sites/genetics , CHO Cells , Carbamates/chemistry , Carbamates/pharmacology , Cricetinae , Cricetulus , Humans , KCNQ Potassium Channels/genetics , KCNQ Potassium Channels/metabolism , KCNQ2 Potassium Channel/genetics , KCNQ3 Potassium Channel/genetics , Membrane Potentials/drug effects , Membrane Potentials/genetics , Membrane Potentials/physiology , Membrane Transport Modulators/chemistry , Molecular Sequence Data , Mutation , Phenylenediamines/chemistry , Phenylenediamines/pharmacology , Pyridines/chemistry , Sequence Alignment , Tryptophan/genetics , Tryptophan/metabolism
9.
Nat Biotechnol ; 24(4): 439-46, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16550174

ABSTRACT

Voltage-gated ion channels regulate many physiological functions and are targets for a number of drugs. Patch-clamp electrophysiology is the standard method for measuring channel activity because it fulfils the requirements for voltage control, repetitive stimulation and high temporal resolution, but it is laborious and costly. Here we report an electro-optical technology and automated instrument, called the electrical stimulation voltage ion probe reader (E-VIPR), that measures the activity of voltage-gated ion channels using extracellular electrical field stimulation and voltage-sensitive fluorescent probes. We demonstrate that E-VIPR can sensitively detect drug potency and mechanism of block on the neuronal human type III voltage-gated sodium channel expressed in human embryonic kidney cells. Results are compared with voltage-clamp and show that E-VIPR provides sensitive and information-rich compound blocking activity. Furthermore, we screened approximately 400 drugs and observed sodium channel-blocking activity for approximately 25% of them, including the antidepressants sertraline (Zoloft) and paroxetine (Paxil).


Subject(s)
Electric Stimulation/methods , Ion Channel Gating/physiology , Membrane Potentials/physiology , Microscopy, Fluorescence/methods , Neurons/physiology , Pharmaceutical Preparations/administration & dosage , Sodium Channels/physiology , Cells, Cultured , Drug Design , Humans , Ion Channel Gating/drug effects , Membrane Potentials/drug effects , Neurons/drug effects , Patch-Clamp Techniques/methods , Sodium Channels/drug effects
10.
Biochem Genet ; 40(11-12): 359-78, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12463345

ABSTRACT

Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the beta-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.


Subject(s)
Cytoskeletal Proteins/genetics , Mitogen-Activated Protein Kinases/genetics , Selection, Genetic , Signal Transduction/genetics , Trans-Activators/genetics , 3T3 Cells , Animals , Biological Assay , Cytoskeletal Proteins/physiology , In Vitro Techniques , Mice , Mitogen-Activated Protein Kinases/physiology , Signal Transduction/physiology , Trans-Activators/physiology , beta Catenin
11.
J Biol Chem ; 277(15): 13219-28, 2002 Apr 12.
Article in English | MEDLINE | ID: mdl-11825900

ABSTRACT

Kvbeta2 binds to K(+) channel alpha subunits from at least two different families (Kv1 and Kv4) and is a member of the aldo-ketoreductase (AKR) superfamily. Proposed functions for this protein in vivo include a chaperone-like role in Kv1 alpha subunit biogenesis and catalytic activity as an AKR oxidoreductase. To investigate the in vivo function of Kvbeta2, Kvbeta2-null and point mutant (Y90F) mice were generated through gene targeting in embryonic stem cells. In Kvbeta2-null mice, Kv1.1 and Kv1.2 localize normally in cerebellar basket cell terminals and the juxtaparanodal region of myelinated nerves. Moreover, normal glycosylation patterns are observed for Kv1.1 and Kv1.2 in whole brain lysates. Thus, loss of the chaperone-like activity does not appear to account for the phenotype of Kvbeta2-null mice, which include reduced life spans, occasional seizures, and cold swim-induced tremors similar to that observed in Kv1.1-null mice. Mice expressing Kvbeta2, mutated at a site (Y90F) that abolishes AKR-like catalytic activity in other family members, have no overt phenotype. We conclude that Kvbeta2 contributes to regulation of excitability in vivo, although not directly through either chaperone-like or typical AKR catalytic activity. Rather, Kvbeta2 relies upon as yet unidentified mechanisms in the regulation of K(+) channel and/or oxidoreductive functions.


Subject(s)
Potassium Channels/physiology , Animals , Base Sequence , Behavior, Animal , Blotting, Western , Cell Line , Cerebellum/metabolism , DNA , Fluorescent Antibody Technique , Mice , Mice, Knockout , Point Mutation , Potassium Channels/genetics , Potassium Channels/metabolism , Sciatic Nerve/metabolism , Xenopus
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