Lead optimization of an acylhydrazone scaffold possessing antiviral activity against Lassa virus

Burgeson, James R.; Gharaibeh, Dima N.; Moore, Ann M.; Larson, Ryan A.; Amberg, Sean M.; Bolken, Tove’ C.; Hruby, Dennis E.; Dai, Dongcheng

doi:10.1016/j.bmcl.2013.08.103

Cited by 20 publications

(15 citation statements)

References 15 publications

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“…We demonstrated that phenothrin has activity against MOPV, GTOV, and CHPV, with IC 50 values lower than 10 μM. The structure of phenothrin is similar to that of the LASV specific entry inhibitor ST-161, which uses a cyclopropyl N -acylhydrazone as a scaffold (26,44). In our study, resistant viruses for LASV or GTOV were not successfully generated in the presence of phenothrin, suggesting this drug is less prone to induce adaptive mutations in the glycoprotein.…”

Section: Discussionmentioning

confidence: 81%

“…Lacidipine is a lipophilic dihydropyridine calcium antagonist. Since calcium channels proved to be a therapeutic target for other enveloped viruses and calcium inhibitors showed promising effects on the entry of the closely-related JUNV and EBOV (22,42-44), we investigated whether lacidipine inhibits LASV entry by acting as a calcium inhibitor. To address this, we first reviewed all 22 calcium inhibitors included in the current FDA drug library; results showed that in addition to lacidipine, only two calcium inhibitors moderately inhibited LASV entry, including diltiazem and cilnidipine.…”

Section: Discussionmentioning

confidence: 99%

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Screening of FDA-approved Drugs and Identification of Novel Lassa Virus Entry Inhibitors

Wang

et al. 2018

Preprint

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12 phenothrin, membrane fusion 13 Word Count (Abstract): 221 14 Word Count (Text): 4,034 ABSTRACT Lassa virus (LASV) belongs to the Mammarenavirus genus (family 22 Arenaviridae) and causes severe hemorrhagic fever in humans. At present, there are 23 no Food and Drug Administration (FDA)-approved drugs or vaccines specific for 24 LASV. Herein, high-throughput screening of an FDA-approved drug library was 25 performed against LASV entry using a pseudo-type virus enveloping LASV 26 glycoproteins. Two hit drugs, lacidipine and phenothrin, were identified as LASV 27 entry inhibitors in the micromolar range. A mechanistic study revealed that both drugs 28 inhibited LASV entry by blocking low-pH-induced membrane fusion. Moreover, 29 lacidipine irreversibly bound to the LASV glycoprotein complex (GPC), resulting in 30 virucidal activity. Adaptive mutant analyses demonstrated that replacement of T40, 31 located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred 32LASV resistance to lacidipine without apparent loss of the viral growth profile. 33Furthermore, lacidipine showed antiviral activity and specificity against both LASV 34 and the Guanarito virus (GTOV), which is also a category A new world arenavirus. 35Drug-resistant variants indicate that the V36M in ectodomain of SSP mutant and 36 V436A in the transmembrane domain of GP2 mutant conferred GTOV resistance to 37 lacidipine, suggesting that lacidipine might act via a novel mechanism other than 38 calcium inhibition. This study shows that both lacidipine and phenothrin are 39 candidates for LASV therapy, and the membrane-proximal external region of the GPC 40 might provide an entry-targeted platform for inhibitors. 41 42 IMPORTANCE Currently, there is no approved therapy to treat Lassa fever; 43 3 therefore, repurposing of approved drugs will accelerate the development of a 44 therapeutic stratagem. In this study, we screened an FDA-approved library of drugs 45 and identified two drugs, lacidipine and phenothrin, which inhibit Lassa virus entry by 46 blocking low-pH-induced membrane fusion. Additionally, both drugs extended their 47 inhibition against the entry of Guanarito virus, and the viral targets of lacidipine were 48 identified. 49 50 Lassa virus (LASV) is an enveloped, negative-sense, bi-segmented RNA virus 51 belonging to the Mammarenavirus genus (family Arenaviridae) (1). 52 Mammarenaviruses consist of 35 unique species currently recognized by the 53 International Committee on Taxonomy of Viruses. The original classification of 54 mammarenaviruses, based mainly on virus antigenic properties; serological, genetic, 55 and geographical relationships; and the rodent host, divided them into new world 56 (NW) and old world (OW) mammarenaviruses (2). The OW Lassa virus and some 57 NW mammarenaviruses, including the Junín virus (JUNV), Machupo virus (MACV), 58 Guanarito virus (GTOV), and Sabiá virus (SABV), are known to cause severe 59 hemorrhagic fever and are listed as biosafety level (BSL) 4 agents (3, 4). LASV 60 infections caus...

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Section: Discussionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Screening of FDA-approved Drugs and Identification of Novel Lassa Virus Entry Inhibitors

Wang

et al. 2018

Preprint

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“…The advent of arenavirus reverse genetics and recombinant pseudotype platforms allowed successful screening of small molecule libraries for entry inhibitors. These screens identified a panel of potent direct antiviral agents that target predominantly viral entry and show distinct specificities among arenaviruses (71,(75)(76)(77)(78)(79)(80)(81). A complementary approach involves the identification of cellular factors required for productive viral entry and their evaluation as antiviral drug targets.…”

Section: Discussionmentioning

confidence: 99%

Identification of Clotrimazole Derivatives as Specific Inhibitors of Arenavirus Fusion

Torriani

Trofimenko

Mayor

et al. 2019

J Virol

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Arenaviruses are a large family of emerging enveloped negative-strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human-pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of “druggable” targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the antiarenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel antiarenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole derivatives for therapeutic intervention against human-pathogenic arenaviruses. IMPORTANCE Emerging human-pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel antiarenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotrimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo. Our study provides the basis for further evaluation of clotrimazole derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate repurposing for therapeutic intervention against human-pathogenic arenaviruses.

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“…4). Furthermore, photoaddition was also blocked by the addition of excess ST-161 and ST-193, two independently identified SIGA fusion inhibitors that are respectively selective for LASV (33) or broadly active against NW and OW arenaviruses (32) (Fig. 6A).…”

Section: Figmentioning

confidence: 99%

“…High-throughput screening campaigns at SIGA Technologies (31)(32)(33)(34) and the Scripps Research Institute (35,36) have recently identified six chemically distinct classes of small-molecule compounds that specifically antagonize pH-induced activation of GPC membrane fusion to prevent arenavirus entry into its host cell (37,38). Several of these inhibitors have been shown to protect against lethal arenavirus infection in small-animal models (31,39).…”

mentioning

confidence: 99%

Small-Molecule Fusion Inhibitors Bind the pH-Sensing Stable Signal Peptide-GP2 Subunit Interface of the Lassa Virus Envelope Glycoprotein

Shankar

Whitby

Casquilho-Gray

et al. 2016

J Virol

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Arenavirus species are responsible for severe life-threatening hemorrhagic fevers in western Africa and South America. Without effective antiviral therapies or vaccines, these viruses pose serious public health and biodefense concerns. Chemically distinct small-molecule inhibitors of arenavirus entry have recently been identified and shown to act on the arenavirus envelope glycoprotein (GPC) to prevent membrane fusion. In the tripartite GPC complex, pH-dependent membrane fusion is triggered through a poorly understood interaction between the stable signal peptide (SSP) and the transmembrane fusion subunit GP2, and our genetic studies have suggested that these small-molecule inhibitors act at this interface to antagonize fusion activation. Here, we have designed and synthesized photoaffinity derivatives of the 4-acyl-1,6-dialkylpiperazin-2-one class of fusion inhibitors and demonstrate specific labeling of both the SSP and GP2 subunits in a native-like Lassa virus (LASV) GPC trimer expressed in insect cells. Photoaddition is competed by the parental inhibitor and other chemically distinct compounds active against LASV, but not those specific to New World arenaviruses. These studies provide direct physical evidence that these inhibitors bind at the SSP-GP2 interface. We also find that GPC containing the uncleaved GP1-GP2 precursor is not susceptible to photo-cross-linking, suggesting that proteolytic maturation is accompanied by conformational changes at this site. Detailed mapping of residues modified by the photoaffinity adducts may provide insight to guide the further development of these promising lead compounds as potential therapeutic agents to treat Lassa hemorrhagic fever. IMPORTANCEHemorrhagic fever arenaviruses cause lethal infections in humans and, in the absence of licensed vaccines or specific antiviral therapies, are recognized to pose significant threats to public health and biodefense. Lead small-molecule inhibitors that target the arenavirus envelope glycoprotein (GPC) have recently been identified and shown to block GPC-mediated fusion of the viral and cellular endosomal membranes, thereby preventing virus entry into the host cell. Genetic studies suggest that these inhibitors act through a unique pH-sensing intersubunit interface in GPC, but atomic-level structural information is unavailable. In this report, we utilize novel photoreactive fusion inhibitors and photoaffinity labeling to obtain direct physical evidence for inhibitor binding at this critical interface in Lassa virus GPC. Future identification of modified residues at the inhibitor-binding site will help elucidate the molecular basis for fusion activation and its inhibition and guide the development of effective therapies to treat arenaviral hemorrhagic fevers.A renaviruses are endemic in rodent populations worldwide, and some species can be transmitted to humans and cause severe hemorrhagic fevers with high morbidity and mortality (1, 2). Lassa virus (LASV) is prevalent in western Africa (3) and can be imported to the United...

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Lead optimization of an acylhydrazone scaffold possessing antiviral activity against Lassa virus

Cited by 20 publications

References 15 publications

Screening of FDA-approved Drugs and Identification of Novel Lassa Virus Entry Inhibitors

Screening of FDA-approved Drugs and Identification of Novel Lassa Virus Entry Inhibitors

Identification of Clotrimazole Derivatives as Specific Inhibitors of Arenavirus Fusion

Small-Molecule Fusion Inhibitors Bind the pH-Sensing Stable Signal Peptide-GP2 Subunit Interface of the Lassa Virus Envelope Glycoprotein

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