In response to the current global health emergency posed by the Zika virus (ZIKV) outbreak and its link to microcephaly and other neurological conditions, we performed a drug repurposing screen of ~6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds; we identified compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. A pan-caspase inhibitor, emricasan, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. Our results demonstrate the efficacy of this screening strategy and identify lead compounds for anti-ZIKV drug development.
ZIKV, a mosquito-borne flavivirus, has spread across the Western Hemisphere in the past year. First isolated in 1947 from a sentinel rhesus macaque in the Ziika Forest region of Uganda1, ZIKV remained in relative obscurity for many years until outbreaks occurred in the Pacific islands and then in the Americas in the past decade. A large outbreak started in Brazil in late 2014, and it is a growing public health concern2. Active transmission has been reported in approximately 60 countries and territories globally. Most human infections are transmitted by mosquitos; however, ZIKV can also spread directly through sexual contact3–6 and vertically from mother to fetus7–9. About 20% of ZIKV-infected individuals develop symptoms, which mostly resemble those caused by other arboviruses, such as dengue viruses or chikungunya virus. Unlike these viruses, however, ZIKV causes congenital defects, including microcephaly10,11, and is associated with Guillain–Barré syndrome, meningoencephalitis and myelitis in infected adults8,11–14.
Since the recent declaration by the World Health Organization (WHO) that ZIKV is a global health concern, rapid progress has been made to understand its pathogenesis and to develop human in vitro models and animal in vivo models15–23. Following clinical observations of ZIKV in fetal brains obtained from infected women9,10, we reported that ZIKV efficiently targets human neural progenitor cells (hNPCs) and attenuates their growth15. This finding provides a potential mechanism for ZIKV-induced microcephaly as hNPCs drive the development of the cortex in the human brain.
Furthermore, we and others have shown that ZIKV infection of brain organoids, which are used as three-dimensional (3D) cellular models of early human brain development, leads to reduced thickness of hNPC and neuronal layers, and an overall reduction in organoid size16,17,20,24, observations that are again consistent with features of microcephaly. These results have also been recapitulated in mouse models20,21,23. Despite these advancements in understanding how ZIKV causes developmental abnormalities and in preclinical studies that are underway to develop vaccines25,26, there is currently no drug approved to treat or prevent ZIKV infection. Drug repurposing screens have recently emerged as an alternative approach to accelerate drug development27,28. Following a repurposing phenotypic screen, new indications for existing drugs may be rapidly identified, and clinical trials can be carried out quickly, which is especially critical for rapidly spreading infectious diseases. For example, recent drug repurposing screens have led to discoveries of potential new candidate therapies for Ebola virus disease29,30, giardiasis31, Entamoeba histolytica infection32, malaria gametocytes33, Exserohilum rostratum infection34, hepatitis C virus (HCV) infection35 and, very recently, ZIKV infection36. On the basis of our previous finding that ZIKV infection of hNPCs results in an increase in caspase-3 activation, followed by cell death15, we designed a compound-screening approach using caspase-3 activity as the primary screening assay, with a secondary cell viability assay for confirmation (Supplementary Fig. 1a). We identified two classes of effective compounds that are capable of protecting neural cells from ZIKV-induced cell death, one consisting of compounds that are antiviral in nature and the other of compounds that are neuroprotective in nature.
Development of high-throughput compound-screening approaches We first quantified caspase-3 activity and cell viability in hNPCs and astrocytes that were derived from human induced pluripotent stem cells (iPSCs), as well as in glioblastoma SNB-19 cells, after ZIKV infection in a 1,536-well plate format (Supplementary Tables 1 and 2). The prototypic ZIKV strain, MR766, was used in the primary screen because it produced the strongest cell death signal in cell culture experiments. The signal-tobasal (S/B) ratios and the coefficients of variation (CV) obtained in the caspase-3 activity assay after a 6-h ZIKV exposure were 2.1-fold and 7.0% for hNPCs, 7.0-fold and 5.9% for SNB-19 cells and 11.0-fold and 9.1% for astrocytes (Supplementary Fig. 1b). The Zʹ factors, which are a measure of statistical effect size and an index for assay quality control37, for hNPCs, SNB-19 and astrocytes were 0.20, 0.68 and 0.72, respectively. Because a Zʹ factor >0.5 indicates a robust screening assay37, the caspase assay using SNB-19 cells or astrocytes is suitable for high-throughput screening. To measure cell viability, we performed an ATP-content assay following ZIKV infection for 3 d (Supplementary Table 2). Cell viability was reduced by 39%, 82% and 69% in hNPCs, SNB-19 cells and human astrocytes, respectively (Supplementary Fig. 1c). The Zʹ factors in these three cell types were 0.06, 0.37 and 0.32, respectively. These results indicated that measuring caspase-3 activity is a better assay for high-throughput compound screening than the cell-viability assay. High-throughput screen of compound collections We carried out a screening campaign, using the caspase-3
activity assay and SNB-19 cells, with the Library of Pharmacologically Active Compounds (LOPAC, 1,280 compounds), the National Center for Advancing Translational Sciences (NCATS ) Pharmaceutical Collection of approved drugs (2,816 compounds) and a collection of clinical candidate compounds (2,000 compounds). Primary hits included a total of 116 compounds that suppressed ZIKV-induced caspase-3 activity in SNB-19 cells. We also carried out an independent primary screen in hNPCs using the same libraries. This second screen resulted in 173 primary hits that included all 116 compounds from the initial caspase-3 screen in SNB-19 cells. All of the results from the primary screen with the approved drug collections, and hit confirmation, were deposited into the open-access PubChem database (https://pubchem.ncbi.nlm.nih.gov/).
Next, the activity of these primary hits from the caspase-3 activity assay was re-evaluated in ZIKV-infected SNB-19 cells, hNPCs and astrocytes, and, notably, was performed in parallel with the compound cytotoxicity assay (Supplementary Fig. 1d,e and Supplementary Table 3). Cytotoxic compounds were then eliminated from the confirmedhit list. Consistent with the screening design, we identified compounds that reduced virally induced caspase activation and apoptosis by either directly preventing ZIKV-induced cell death or suppressing ZIKV replication (Supplementary Table 4). Protection from ZIKV-induced cell death by emricasan Emricasan, a pan-caspase inhibitor, was identified as the most potent anti-cell-death compound, with half-maximal inhibitory concentration (IC50) values of 0.13–0.9 μM in both caspase activity and cell viability assays for SNB-19 cells against three ZIKV strains: MR766 (1947 Ugandan strain), FSS13025 (2010 Cambodian strain) and PRVABC59 (2015 Puerto Rican strain) (Fig. 1a). It was also effective for all three cell types tested (Supplementary Fig. 1f and Supplementary Table 5). In addition, emricasan treatment reduced the number of active (cleaved) caspase-3-expressing
forebrain-specific hNPCs after infection with FSS13025, in both monolayer and 3D organoid cultures (Fig. 1b,c). Emricasan treatment of ZIKV-exposed brain organoids did not seem to affect hNPC proliferation, as compared to the mock-treated organoids, when evaluated by phosphohistone3 (PH3) expression (106 ± 10%; n = 8; P = 0.7; one-way analysis of variance (ANOVA)). Notably, ZIKV antigen persisted in both 2D and 3D cultures after emricasan treatment (Fig. 1b,c). Therefore, emricasan shows neuroprotective activity for hNPCs but does not suppress ZIKV replication.
Identification of antiviral compounds against ZIKV
Using expression of ZIKV protein NS1 as a read-out to screen anti-ZIKV activity, we identified two compounds from the primary hit list that substantially inhibited ZIKV infection of SNB-19 cells (Supplementary Fig. 2a). The first was niclosamide, a US Food and Drug Administration (FDA)-approved drug for treating worm infections in both humans and domestic livestock; the other was PHA-690509, an investigational compound that functions as a cyclin-dependent kinase inhibitor (CDKi). Both compounds inhibited replication of all three strains of ZIKV, as measured by NS1 expression, in a dose-dependent manner (Fig. 2a–d and Supplementary Fig. 2b,c). We further validated the antiviral activity of these two compounds using independent approaches. First, measurement of intracellular ZIKV RNA levels showed IC50 values of 1.72 μM and 0.37 μM for PHA-690509 and niclosamide, respectively (Supplementary Fig. 2d,e). Second, both compounds suppressed production of infectious ZIKV particles at submicromolar concentrations (Fig. 2e). To investigate the underlying cellular mechanism, we performed time-of-addition experiments in SNB-19 cells (Fig. 3a). Both compounds effectively inhibited ZIKV infection when added either 1 h before or 4 h after virus inoculation (Fig. 3b). In contrast, a monoclonal antibody to AXL, a putative ZIKV entry factor38,39, was only effective when added before inoculation (Fig.3b). Furthermore, the reduction of ZIKV RNA by treatment with these compounds was apparent only after the entry phase (0–4 h after infection) and correlated with the replication phase (4–24 h) of the infection cycle (Supplementary Fig. 3). Taken together, these results indicate that niclosamide and PHA-690509 inhibit ZIKV infection at a post-entry stage, probably at the viral RNA replication step. Pharmacological CDKis have been shown to inhibit replication of diverse viruses in culture, including herpes viruses and HIV40–45, and depletion of CDK9 impairs influenza A viral replication46. The possibility of ZIKV inhibition by a CDKi is particularly intriguing, given our recent observation that ZIKV infection and cell cycle regulation are intimately connected15. We therefore tested 27 additional structurally distinct CDKis for inhibition of ZIKV infection (Supplementary Table 6). We identified nine effective compounds using SNB-19 cells (Fig. 3c,d and Supplementary Fig. 4a), whereas four non-CDK-kinase inhibitors showed minimal anti-ZIKV activity (Supplementary Fig. 4b). Analyses of ZIKV production showed IC50 values at submicromolar concentrations for nine of the ten CDKis tested, with seliciclib and RGB-286147 at 24 nM and 27 nM, respectively (Fig. 3d). Using the clinical isolate from the 2015 Puerto Rico Zika outbreak, PRVABC59, we next examined the effectiveness of the identified compounds in hNPCs and astrocytes, both of which are target cells for ZIKV infection in the fetal brain9. Niclosamide and PHA-690509 treatments inhibited ZIKV infection and production in these central nervous system cells (Fig. 4a–c). ZIKV targets astrocytes in the adult mouse brain47. Analysis of ZIKV production after infection of human astrocytes showed IC50 values of ~0.2 mM for both niclosamide and PHA-690509 (Fig. 4c). Quantification of astrocyte viability showed minimal toxicity for niclosamide, PHA-690509 and four CDKis at levels <3 mM (Supplementary Fig. 5a). Given the critical role of CDKs in cell cycle regulation, we examined the effect of PHA-690509 and seliciclib, the most potent CDKi for ZIKV inhibition (Fig. 3d), on hNPC proliferation. ZIKV infection led to a drastic reduction in hNPC proliferation, which was partially rescued by treatment with either compound (Supplementary Fig.5b). Furthermore, treatment with PHA-690509 (1 mM) or seliciclib (5 mM) alone had minimal effect on hNPC proliferation in brain organoids.
Benefit of combining neuroprotective and antiviral compounds
Finally we explored combining these two categories of compounds (neuroprotective and antiviral). The two-drug combination of emricasan and PHA-690509 showed an additive effect in inhibiting caspase- 3 activity in SNB-19 cells (Fig. 4d). A similar additive effect was found to preserve astrocyte viability after ZIKV infection (Fig. 4). Notably, emricasan did not interfere with PHA-690509’s ability to inhibit ZIKV infection in the combination treatment (Fig. 4b and Supplementary Fig. 6a–c). We also tested the effect of sequential treatment with the two categories of compounds. We found that treatment of PRVABC59-infected hNPCs with emricasan for 72 h followed by niclosamide treatment for 48 h led to the recovery of ZIKV-negative hNPCs, suggesting a beneficial effect of ‘buying time’ by inhibiting apoptosis to allow the infected cells to recover.
Here we developed two ultra-high-throughput assays, using human neural cells, to measure ZIKV-induced caspase-3 activity and cell viability, and we screened over 6,000 approved drugs and drug candidate compounds. Our efforts so far have led to the identification of small molecules that either protect against cell death in multiple neural cell types or suppress ZIKV replication. Emricasan, also called IDN-6556 or PF-03491390, is an inhibitor of activated caspases, and it has sub- to nanomolar activity in vitro48,49. Emricasan is currently being evaluated in phase 2 clinical trials for the reduction of hepatic injury and liver fibrosis caused by chronic HCV infection50,51. Emricasan was well tolerated in human trials, and there were no significant adverse events51. It was reported that overall and maximum concentrations of emricasan (orally, twice daily, ×4 d) in human blood were 1.90 mg/ml (3.35 mM) and 2.36 mg/ml (4.15 mM), respectively. Therefore, the reported human plasma concentration of emricasan is about tenfold higher than the IC50 for inhibition of increased caspase-3 activity and cell death caused by ZIKV infection in vitro. Future animal studies will be critical to evaluate the efficacy of emricasan in vivo. Whether it is safe to use emricasan during pregnancy for ZIKV infection in humans will need to be evaluated in preclinical toxicology studies and in clinical trials.
Ten structurally unrelated CDKis inhibit ZIKV replication and production, supporting a role for CDKs in the life cycle of ZIKV in human cells. Because flaviviruses are not known to encode any CDKs, these results suggest that one or more cellular CDKs in the host may be important for ZIKV replication. Further studies on target identification, including targeted kinome siRNA or CRISPR screens, may reveal additional insights into the mechanism of action for these inhibitors. Although many CDKis are being evaluated in clinical trials for various cancers, cystic fibrosis and Cushings disease, these compounds may not be suitable for pregnant women because of potentially hazardous effects on the fetus52. In our in vitro analyses, treatment with PHA-690509 or seliciclib partially rescued the ZIKV-induced reduction of hNPC proliferation, and the treatment with either compound in the absence of ZIKV infection showed a minimal effect on hNPC proliferation in brain organoid cultures, which model early human brain development in vitro (Supplementary Fig. 5c,d). Future studies in animal models, including both rodents and primates, are critical to test the efficacy and toxicity of identified compounds in vivo. Notably, these CDKis and their derivatives may be useful for treating nonpregnant humans who face an increased risk of Guillain–Barré syndrome and other conditions following ZIKV infection. For example, viral RNA and infectious virus have been detected in the semen of men months after acute symptoms have resolved53. In addition, these CDKis will be valuable chemical tools in probing the roles of specific types of CDKs in ZIKV infection, which could serve as new targets for anti-ZIKV drug development. The structural diversity of effective CDKis also provides multiple scaffolds for lead-compound optimization using medicinal chemistry. Niclosamide is an FDA-approved drug (trade name Niclocide) that has been used in humans to treat worm infections for nearly 50 years, and it is well tolerated54,55. It is known to inhibit several viruses in culture systems, including the Japanese encephalitis flavivirus56–58. Its broad antiviral activity has been attributed to its ability to neutralize endolysosomal pH and interfere with pH-dependent membrane fusion57, which is an essential step in the common virus entry pathway. Our time-of-addition and time-course analyses, however, suggest that inhibition by niclosamide occurs at a post-entry step, such as replication. Future molecular studies of its mechanism of action may provide additional targets for drug development. Niclosamide is a category B drug, which indicates that no risk to fetuses has been found in animal studies. It has low toxicity in mammals with an oral median lethal dose (LD50) value of 5,000 mg per kg body weight (mg/kg) in rats59. The potency of niclosamide on inhibition of ZIKV replication is in the submicromolar range, whereas clinically it can be delivered at micromolar levels59. Additionally, a pro-drug formulation may further increase the bio-availability of this compound, which is currently indicated for treating intestinal parasites. The WHO recommends that niclosamide may be used during pregnancy because it has not been shown to be mutagenic, teratogenic or embryotoxic (http://apps.who.int/medicinedocs/en/d/Jh2922e/3.1.3.html). The US Centers for Disease Control and Prevention (CDC) concurs with the WHO recommendation and further recommends that “for individual patients in clinical settings, the risk of treatment (with Niclosamide) in pregnant women who are known to have an infection needs to be balanced with the risk of disease progression in the absence of treatment” (http://www.cdc.gov/parasites/hymenolepis/health_professionals/). Independently of evaluating the potential benefits and risks for pregnant women, niclosamide could be used to reduce viral load in infected men and nonpregnant women, which could reduce transmission and potentially prevent Guillain–Barré syndrome and other ZIKV-related complications in humans. Despite rapid progress in the preclinical development of anti-ZIKV vaccines25,26, testing the safety and efficacy of vaccines in humans can take a substantial amount of time. Effective countermeasures against ZIKV, including small-molecule therapeutics, are also urgently needed. Our findings and the tools provided here should significantly advance current ZIKV research and have an immediate effect on the development of anti-ZIKV therapeutics. Furthermore, our findings could have implications for combating infections by other arboviruses, such as dengue viruses, chikungunya virus and West Nile virus, many of which can cause devastating illness.