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Structural and mechanistic insight from antiviral and antiparasitic enzyme drug targets for tropical infectious diseases
Available online at www.sciencedirect.com
ScienceDirect Structural and mechanistic insight from antiviral and antiparasitic enzyme drug targets for tropical infectious diseases Andre Schutzer de Godoy, Rafaela Sachetto Fernandes, Anna Caroline Campos Aguiar, Renata Vieira Bueno, Nathalya Cristina de Moraes Roso Mesquita, Rafael Victorio Carvalho Guido and Glaucius Oliva With almost half of the world population living at risk, tropical infectious diseases cause millions of deaths every year in developing countries. Considering the lack of economic prospects for investment in this field, approaches aiming the rational design of compounds, such as structure-based drug discovery (SBDD), fragment screening, target-based drug discovery, and drug repurposing are of special interest. Herein, we focused in the advances on the field of SBDD targeting arboviruses such as dengue, yellow fever, zika and chikungunya enzymes of the RNA replication complex (RC) and enzymes involved in a variety of pathways essential to ensure parasitic survival in the host, for malaria, Chagas e leishmaniasis diseases. We also highlighted successful examples such as promising new inhibitors and molecules already in preclinical/clinical phase tests, major gaps in the field and perspectives for the future of drug design for tropical diseases. Address Institute of Physics of Sa˜o Carlos, University of Sa˜o Paulo, Av. Joao Dagnone, 1100 - Jardim Santa Angelina, Sa˜o Carlos 13563-120, Brazil Corresponding author: Oliva, Glaucius ([email protected])
Current Opinion in Structural Biology 2019, 59:65–72 This review comes from a themed issue on Catalysis and regulation
Africa, South and Central Americas and East Asia [1,2]. These places are constantly riddled by spreads of other arthropod-borne diseases, such as leishmaniasis, chagas, yellow fever, different types of encephalitis, and are also on the constant threat of new emerging infectious diseases, such as chikungunya and zika [3]. Although many advances in the development of antiparasitic and antiviral compounds were achieved over the last decade, the majority of the tropical diseases remain without a satisfactory treatment [3]. Despite many scientific and social difficulties of drug discovery for tropical diseases, the major challenge remains the lack of economic prospects for investment in this field. Therefore, approaches aiming the rational design of compounds, such as SBDD, fragment screening, target-based drug discovery, and drug repurposing are of special interest. In this context, enzyme inhibitors offer unique opportunity for drug discovery. Nowadays about half of the known drug targets are enzymes and the rational to design new inhibitors has an established systematic approach [4]. In this review, we point to the advances on the field of SBDD targeting pathogen enzymatic pathways, including successful examples, major gaps on the field and perspectives for the future of drug design for tropical diseases.
Edited by Philip Cole and Andrea Mattevi
Antiviral drug targets https://doi.org/10.1016/j.sbi.2019.02.014 0959-440X/ã 2018 Elsevier Inc. All rights reserved.
Introduction Many infectious diseases are transmitted by arthropod vectors, being of special concern for countries situated at the tropical and subtropical regions. With about 40% of the world population living in risk areas, diseases such as dengue and malaria cause enormous health and economic impact on developing countries, especially in www.sciencedirect.com
Arbovirus, a contraction of ‘arthropod-borne virus’, is a term used to designate those viruses transmitted by insect vectors, usually ticks and mosquitos. Members of the families Flaviviridae, Peribunyaviridae, Phenuiviridae, and Togaviridae are known to cause human and animal diseases in a complex transmission cycle involving the virus, vertebrate host and the vector. Among the members of these families, the flaviviruses Yellow Fever (YFV), Dengue (DENV), Zika (ZIKV), and West Nile (WNV), as well as the togaviruses Chikungunya (CHIKV) and Mayaro (MAYV) are some of the emergent and reemergent arboviruses of public health importance in tropical and subtropical countries [5]. Dengue fever alone affects about 100 million people with clinical symptoms every year, with more than half of the Current Opinion in Structural Biology 2019, 59:65–72
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world population living in risk areas [1]. The constant resurgences of YFV in Africa and South America and the 2015–2016 epidemic outbreak of ZIKV in the Americas are examples of the great impact of flaviviruses in public health [6,7]. CHIKV outbreaks have been reported in Africa, Asia, Europe and America over the past 5 years, causing large epidemics with a higher incidence of more severe forms of the disease than previously reported [8]. Although there are vaccines to prevent YFV infections, specific treatments for any arbovirus-associated diseases are still not available [9]. The flavivirus genome is a positive-sense single-stranded RNA that encodes ten non-redundant proteins: three structural proteins that forms the viral capsid, membrane and envelope, and seven non-structural proteins (NS) that are responsible for the replication of the viral RNA inside the host cell [10]. The N-terminal domain of NS3 is a trypsin-like serine protease (NS3Pro) which requires the cofactor activity provided by the membrane-bound NS2B to process the viral polyprotein into mature proteins [11]. NS3 C-terminal region forms the RNA helicase domain responsible for unwinding the double stranded RNA before elongation [12]. The RNA elongation is performed by the C-terminal domain of NS5, which is an RNAdependent RNA polymerase (RdRp). The nascent positive RNA is capped at the NS5 N-terminal methyltransferase domain. All those enzymatic functions are essential for the viral replication cycle and, therefore, natural targets for antiviral drug discovery [13]. Over the last few years, the development of new inhibitors for flaviviruses targets has significantly grown due to the abundance of structural information for NS3 and NS5, together with well-established enzymatic and cell assays. The most studied protease inhibitors are peptide-derived molecules, such as peptide aldehydes and peptide boronic acids that shown a low micromolar to nanomolar inhibition of DENV serotype 2 (DENV-2) [14], WNV [15,16], and ZIKV NS3Pro [17]. However, potent peptide inhibitors of NS2B–NS3 protease detected in enzymatic assays often fail in cell-based assays, probably due to the low permeability of peptide derived compounds [18]. Yet, considering the success of HIV/AIDS protease inhibitor cocktails, this target remains a promising candidate for the development of broad-spectrum antiviral molecules [3]. The NS3 helicase structures have been reported for many flaviviruses, including DENV-2 [19], YFV [20] and ZIKV [21]. Besides the helicase activity, this enzyme also has an RNA50 triphosphatase activity (RTPase) essential to ensure its function in viral replication. A few inhibitors of the DENV NS3 helicase have been studied, like the benzoxazole analogue ST-610 [22] and a pyrrolone derivative [23]. Nevertheless, the lack of specificity of the RNA and NTP binding sites presumably leads to a significant toxicity as compounds targeting these sites might also bind Current Opinion in Structural Biology 2019, 59:65–72
to similar cellular proteins with helicase/NTPase activities. Still, most of the ligand-target structural information available is restricted to the ATP binding site of NS3 helicase (Figure 1). New structural-based techniques aiming to identify ligands for the RNA cleft are urgently required. The methyltransferase domain of NS5 is a small and globular protein, with a GTP and a S-adenosyl methionine binding pocket [24]. Many analogs of these molecules, such as Sinefugin, exhibited high affinity for the enzyme, but low effect on infected cells due to permeability issues [24]. Although structural information of many ligands is available and SBDD is possible, the accumulated failures in transposing the in vitro to in situ results turned the methyltransferase into a less interesting target for drug discovery [9,24]. In contrast, the RdRp domain of NS5 is probably the most interesting target for SBDD in flaviviruses. With a long and conserved cleft for accommodating the RNA during elongation, the structural data reveal many putative binding sites for SBDD [25]. The first non-nucleoside candidate reported to interact directly with the NS5 was Compound 27, an acyl sulfonamide analogue with potential antiviral activity against all four dengue serotypes, but not much is known about its subsequent development [25]. Other non-nucleoside inhibitors, such as ivermectin or fenretinide, are described to block the NS5 IMP-a/b1 interaction pathway in cells, but still no clinical trials were performed to test their efficacy [24,26]. Perhaps the most important lesson for designing inhibitors for flavivirus comes from another branch of this virus phylogeny. The Hepatitis C virus (HCV) is a member of the Flaviviridae family, in the genus Hepacivirus, and many genomic and structural similarities are shared among the groups. In 2013 a nucleotide analog, denominated sofosbuvir (PSI-7977), was approved for combination treatment of the three serotypes of HCV [27]. This prodrug is a phosphoramidate that is metabolized into a nucleotide analog that blocks RNA elongation in HCV NS5RdRp [27]. The oral administration of sofosbuvir in combination with a NS5A inhibitor, such as ledipasvir, has demonstrated impressive results in the treatment of chronicle HCV infections [28]. The mechanism in which the drug inhibits the HCV Genotype 2A RdRp via blocking RNA elongation was depicted with the ternary crystal structure of the complex (Figure 1) [29]. The methyluridine portion of the drug stacks to the RNA via Watson–Crick pairing, while the fluoride bounds to Asn291, selectively inhibiting the viral replication at the nanomolar range [29,30]. Despite the low sequence identity between the RdRp of HCV and other flavivirus (10–18%), a few residues involved in PSI-7977 stabilization are mostly conserved (Figure 1). Yet, stereochemistry and charge distribution of the relative region are remarkably different, meaning that further optimization may be required for the application of this molecule. Still, recent reports indicate that PSI-7977 might exhibit strong antiviral www.sciencedirect.com
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Figure 1
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(a) Crystal structures of NS5 RdRp-DNA-Sofosbuvir complex from HCV showing details of (a) the mechanism in which the drug blocks RNA elongation by competing with nucleotides and (b) the relative position of Sofosbuvir in Zika virus crystal structure. For both, nucleosides are colored in green and surface is colored according to calculated charge from 5 kV (red) to 5 kV (blue). (c) Superposition of all available NS3 helicases from flavivirus (blue) with ligands show as red spheres, showing the clear lack of structural information the RNA cleft. Subdomains (SD) are depicted with orange dashes, ATP site with purple dashes and RNA cleft is demarked with arrow. (d) Structure details of Dengue virus RdRp in complex with compound 27.
activity against ZIKV, suggesting a broad-spectrum activity for nucleoside analogs [31,32]. The Alphavirus genome consists of a single stranded and positive sense RNA with two open read frames (ORF). The 30 ORF encodes five structural proteins: capsid C, envelope glycoproteins E1, E2, and E3, and the protein 6K. The 50 ORF encodes the four non-structural proteins NSP1, NSP2, NSP3, and NSP4, which the structure association results in the viral replication complex [33]. Apart from the NSP2 protease domain and NSP3 macro domain, wherein structures are currently available at the Protein Data Bank, the alphavirus non-structural proteins NSP1, NSP2 helicase domain and NSP4 have not been elucidated yet. Despite of that, NSP1 and NSP4 are considered attractive molecular targets for alphaviruses, as well as NSP2, and they have been explored by integrating in silico screen strategies and cell-based assays [34,35]. The NSP1 has guanine-7-methyltransferase (MTase) and guanylyltransferase (GTase) activities, directing the methylation and capping of the viral genomic RNA, essential to www.sciencedirect.com
prevent its exposure to host cell exonucleases. Since the non-conventional alphavirus RNA capping mechanism differs from the host cell mechanism by the sequence of reactions, NSP1 has been considered an attractive target to develop selective antiviral drugs [36]. Studies suggests that quinine, an antimalarial drug that reduces CHIKV replication in vitro, affects the NSP1 function [37]. More recently, in vitro screening against CHIKV strains associated with enzymatic assays for Venezuelan equine encephalitis virus (VEEV) suggested a novel class of triazolopyrimidinones as potent CHIKV replication inhibitors. Specifically targeting the guanylylation step catalyzed by NSP1, the lead compound MADTP-372 prevented the cytopathic effect (CPE) induced by CHIKV with an EC50 value of 2.6 mM. These results suggest that MADTP compounds are a promising antiviral therapy for CHIKV infections, although preclinical and clinical studies still need to be performed [38]. Alphavirus NSP2 is a multifunctional protein with a Cterminal protease activity necessary to cleave the Current Opinion in Structural Biology 2019, 59:65–72
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polyprotein NSP1234 into the four mature NSPs. Regardless of the low sequence identity, the NSP2 fold is highly conserved between species and its structure was elucidated for VEEV, CHIKV, and Sindbis virus. Using a NSP2 CHIKV comparative model, approximately 5 million compounds were screened in silico, a hit compound was selected and structural analogs were evaluated in vitro using a CHIKV cell-based assay. Compound 25, a acrylohydrazide derivative, the most potent molecule was shown to inhibit the CHIKV induced CPE with an EC50 of 3.2 mM. [39] Further studies have stablished structure–activity relationships, proposed a binding model for structural analogs in the proteolytic site of CHIKV NSP2 and identified novel and selective CHIKV induced CPE inhibitors at the low micromolar range. Additional structural modifications are ongoing to improve the antiviral activity of those inhibitors [40]. The NSP4 is an RNA-dependent RNA polymerase (RdRp) with well conserved motifs between positivesense RNA viruses. On the basis of that, a CHIKV NSP4 structure was built by comparative modelling
and explored by docking, aiming to investigate the binding mode of sofosbuvir [41]. The interactions observed between sofosbuvir and CHIKV NSP4 are similar to those observed for the natural substrate uridine triphosphate (UTP). Moreover, in vitro assays have shown that sofosbuvir inhibits the CHIKV replication in Huh-7 hepatoma cells and astrocytes derived from induced pluripotent stem cells. Furthermore, it has also been observed a systemic protective effect of sofosbuvir in vivo, which prevents the inflammatory cell increase caused by CHIKV. Altogether, those findings highlight the possibility that genetically distinct viruses such as Togaviridae and Flaviridae members could also be susceptible to sofosbuvir and propose clinical investigations on the use of this drug as a treatment for CHIKV infection [41].
Antiparasitic drug targets The malaria disease is caused by Plasmodium spp. protozoan and remains a significant global health challenge, killing more than 400 000 people each year, the majority being children under 5 years old living in sub-Saharan Africa [2]. The efficacy of antimalarial drugs has
Figure 2
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(a) Structure of Plasmodium falciparum dihydroorotate dehydrogenase. (b) Binding details of the Plasmodium falciparum dihydroorotate dehydrogenase bound to DSM265 (c) Crystal structure of Pfdhfr-ts. (d) Binding details of CYP51 from Trypanosoma cruzi in complex with posaconazole. Current Opinion in Structural Biology 2019, 59:65–72
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Antiviral and antiparasitic enzyme drug targets de Godoy et al. 69
Figure 3
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decreased due to the ongoing resistance of the parasite against clinically available drugs, including the current artemisinin-based combination therapy, which reinforces the extremely urgent need for new antimalarials [2]. www.sciencedirect.com
Over the last decade, many potential antimalarial drugs have been identified by phenotypic assays and subsequently demonstrated to target enzymes involved in hemoglobin hydrolysis, protein synthesis, pyrimidine Current Opinion in Structural Biology 2019, 59:65–72
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biosynthesis, post-translational modifications and mitochondrial metabolism. A recent study tracked multiple genes that are potentially involved in the resistance acquisition by the parasite, including two new transporters and several enzymes as dihydrofolate reductase– thymidylate synthase (Pfdhfr-ts), farnesyltransferase, dipeptidyl aminopeptidase 1, and an aminophospholipid-transporting P-type ATPase [42]. Except for the Pfdhfr-ts (Figure 2), the high-resolution structures of these enzymes are still unknown, preventing the complete understanding of the underpinning molecular mechanisms and further structure-based optimization. A successful example derived from structure-based studies is the DSM265 compound, a triazolopyrimidine class inhibitor, which is currently in phase II clinical trials conducted in patients infected with P. vivax and P. falciparum [43]. Its target, the enzyme Dihydroorotate Dehydrogenase (DHODH), is a flavin (FMN)dependent mitochondrial enzyme, involved in a key step of the nucleic acid synthesis pathway (Figure 2). Unlike humans, Plasmodium relies entirely on the de novo pathway to acquire pyrimidines for DNA and RNA synthesis. DHODH is composed of a core b/a-barrel with an Nterminal a-helix that interacts with the mitochondrial membrane allowing the binding of Coenzyme Q10 (CoQ). The DSM265 inhibitor-binding pocket sits between FMN and the N-terminal a-helix, likely to be the binding-site for CoQ (Figure 2). This pocket is primarily hydrophobic and only two hydrogen bonds are formed between DSM265 and the protein via Arg-265 and His-185. The amino acid composition of the binding-site is highly variable between the Plasmodium and human enzymes and this property is thought to underlie the strong selectivity for the parasite over human DHODH [44]. Another compound currently in phase II clinical trial is MMV390048, a 2-aminopyridine derivative, which targets the phosphatidylinositol 4-kinase (PI4K), an enzyme that phosphorylates lipids to regulate intracellular signaling and trafficking in Plasmodium spp. The inhibitor is efficacious in a single dose and acts in all life cycle stages of the parasite [45]. Phase I compounds include P218, an inhibitor of dihydrofolate reductase; SJ733, a PfATP4 inhibitor; and M5717, an inhibitor of translation elongation factor 2, essential for protein synthesis [46,47]. Chagas disease, caused by the kinetoplastid parasite Trypanosoma cruzi, affects 6–7 million people worldwide, killing about 10 000 people annually. The disease is endemic in Latin America and the current treatment is limited to two rather old and suboptimal nitroheterocyclic drugs, benznidazole and nifurtimox. Significant efforts have been made to repurpose antifungal azole drugs targeting sterol biosynthesis, like CYP51, one of the most studied molecules, to treat Chagas disease Current Opinion in Structural Biology 2019, 59:65–72
(Figure 2). However, in controlled clinical trials, two antifungals, posaconazole (Noxafil, Merck) and ravuconazole (E1224, Eisai, Tokyo) showed inferior potency and side effects in comparison with the current treatment with benznidazole [48]. A similar case happens with visceral leishmaniasis, caused by the Leishmania spp. The disease causes about 40 000 deaths per year, and the available treatments are far from ideal with high cost, toxicity, and without clear mechanism of actions determined. A new promising lead molecule, GSK3186899/DDD853651, a pyrazolopyrimidine derivative, apparently acts by inhibiting the parasite cdc-2 related kinase 12, and showed great efficiency in mouse models. The efficiency of these compounds in clinical trials is yet to be tested [49]. Another bicyclic nitroimidazole molecule (DNDI-0690) showing excellent activity in vitro against both visceral and cutaneous leishhave progressed toward preclinical maniasis, development and a decision to progress to Phase I single ascending dose in healthy volunteers was agreed in 2018 [50].
Conclusion Despite many advances in the drug discovery field, most tropical diseases remain without a satisfactory treatment and our understanding of the interactions between host and pathogen is quite limited. Here, we highlighted some potential new inhibitors of enzymatic pathways of viral and parasitic pathogens of public health importance (Figure 3). There are still major gaps in this field, including the lack of structural information and mechanism of actions validated. Nevertheless, the accumulated knowledge from phenotypic screens in combination with biophysical and computational methods and the development of new sequencing and genome modification techniques allowed the discovery of many new putative targets for drug discovery.
Conflict of interest statement Nothing declared.
Acknowledgements The authors would like to thank the funding from the Sao Paulo Research Foundation – FAPESP (CEPID grant # 2013/07600-3 and post-doctoral grants 2015/18192-9, 2016/19712-9, 2018/05130-3), The National Council for Scientific and Technological Development (grants 405330/2016-2, 307132/2016-1, 151058/2018-0) and the Instituto Serrapilheira (#grant Serra1708-16250).
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38. Delang L, Li C, Tas A, Que´rat G, Albulescu IC, De Burghgraeve T, Segura Guerrero NA, Gigante A, Piorkowski G, Decroly E et al.: The viral capping enzyme nsP1: a novel target for the inhibition of chikungunya virus infection. Sci Rep 2016, 6:1-10. 39. Bassetto M, De Burghgraeve T, Delang L, Massarotti A, Coluccia A, Zonta N, Gatti V, Colombano G, Sorba G, Silvestri R et al.: Computer-aided identification, design and synthesis of a novel series of compounds with selective antiviral activity against chikungunya virus. Antiviral Res 2013, 98:12-18. 40. Tardugno R, Giancotti G, De Burghgraeve T, Delang L, Neyts J, Leyssen P, Brancale A, Bassetto M: Design, synthesis and evaluation against chikungunya virus of novel small-molecule antiviral agents. Bioorganic Med Chem 2018, 26:869-874. 41. Ferreira AC, Reis PA, de Freitas CS, Sacramento CQ, Villas Boˆas Hoelz L, Bastos MM, Mattos M, Rocha N, Gomes de Azevedo Quintanilha I, da Silva Gouveia Pedrosa C et al.: Beyond members of the flaviviridae family, sofosbuvir also inhibits chikungunya virus replication. Antimicrob Agents Chemother 2018, 63: e01389-e01418. Article suggesting sofosbuvir might be active against Chikungunya virus, including in vivo assays. 42. Cowell A, Winzeler E: Exploration of the Plasmodium falciparum resistome and druggable genome reveals new mechanisms of drug resistance and antimalarial targets. Microbiol insights 2018, 11 http://dx.doi.org/10.1177/1178636118808529. Article mapping putative genes involved in drug resistance acquisition from the Plasmodium. 43. Llanos-Cuentas A, Casapia M, Chuquiyauri R, Hinojosa JC, Kerr N, Rosario M, Toovey S, Arch RH, Phillips MA, Rozenberg FD et al.: Antimalarial activity of single-dose DSM265, a novel plasmodium dihydroorotate dehydrogenase inhibitor, in patients with uncomplicated Plasmodium falciparum or Plasmodium vivax malaria infection: a proof-of-concept, open-label, phase 2a study. Lancet Infect Dis 2018, 18:874-883.
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44. Phillips MA, Lotharius J, Marsh K, White J, Dayan A, White KL, Njoroge JW, El Mazouni F, Lao Y, Kokkonda S et al.: A longduration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria. Sci Transl Med 2015, 7:296ra111. Article describing the development and target validation for the compound DSM265, including biological activity, pharmacological properties and preclinical trials. 45. Paquet T, Le Manach C, Cabrera DG, Younis Y, Henrich PP, Abraham TS, Lee MCS, Basak R, Ghidelli-Disse S, LafuenteMonasterio MJ et al.: Antimalarial efficacy of MMV390048, an inhibitor of plasmodium phosphatidylinositol 4-kinase. Sci Transl Med 2017, 9:eaad9735. Article describing the development and target validation for the compound MMV390048. 46. Safety, Tolerability and Chemoprotective Activity of P218 in PfSPZ Challenge Model - ClinicalTrials.gov. Accessed 15 February 2019. 47. Baragan˜a B, Hallyburton I, Lee MCS, Norcross NR, Grimaldi R, Otto TD, Proto WR, Blagborough AM, Meister S, Wirjanata G et al.: A novel multiple-stage antimalarial agent that inhibits protein synthesis. Nature 2015, 522:315-320. 48. Morillo CA, Waskin H, Sosa-Estani S, del Carmen Bangher M, Cuneo C, Milesi R, Mallagray M, Apt W, Beloscar J, Gascon J et al.: Benznidazole and posaconazole in eliminating parasites in asymptomatic T. Cruzi carriers. J Am Coll Cardiol 2017, 69:939-947. 49. Wyllie S, Thomas M, Patterson S, Crouch S, De Rycker M, Lowe R, Gresham S, Urbaniak MD, Otto TD, Stojanovski L et al.: Cyclindependent kinase 12 is a drug target for visceral leishmaniasis. Nature 2018, 560:192-197. 50. Petri e Silva SCS, Palace-Berl F, Tavares LC, Soares SRC, Lindoso JAL: Effects of nitro-heterocyclic derivatives against Leishmania (Leishmania) infantum promastigotes and intracellular amastigotes. Exp Parasitol 2016, 163:68-75.
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ScienceDirect Structural and mechanistic insight from antiviral and antiparasitic enzyme drug targets for tropical infectious diseases Andre Schutzer de Godoy, Rafaela Sachetto Fernandes, Anna Caroline Campos Aguiar, Renata Vieira Bueno, Nathalya Cristina de Moraes Roso Mesquita, Rafael Victorio Carvalho Guido and Glaucius Oliva With almost half of the world population living at risk, tropical infectious diseases cause millions of deaths every year in developing countries. Considering the lack of economic prospects for investment in this field, approaches aiming the rational design of compounds, such as structure-based drug discovery (SBDD), fragment screening, target-based drug discovery, and drug repurposing are of special interest. Herein, we focused in the advances on the field of SBDD targeting arboviruses such as dengue, yellow fever, zika and chikungunya enzymes of the RNA replication complex (RC) and enzymes involved in a variety of pathways essential to ensure parasitic survival in the host, for malaria, Chagas e leishmaniasis diseases. We also highlighted successful examples such as promising new inhibitors and molecules already in preclinical/clinical phase tests, major gaps in the field and perspectives for the future of drug design for tropical diseases. Address Institute of Physics of Sa˜o Carlos, University of Sa˜o Paulo, Av. Joao Dagnone, 1100 - Jardim Santa Angelina, Sa˜o Carlos 13563-120, Brazil Corresponding author: Oliva, Glaucius ([email protected])
Current Opinion in Structural Biology 2019, 59:65–72 This review comes from a themed issue on Catalysis and regulation
Africa, South and Central Americas and East Asia [1,2]. These places are constantly riddled by spreads of other arthropod-borne diseases, such as leishmaniasis, chagas, yellow fever, different types of encephalitis, and are also on the constant threat of new emerging infectious diseases, such as chikungunya and zika [3]. Although many advances in the development of antiparasitic and antiviral compounds were achieved over the last decade, the majority of the tropical diseases remain without a satisfactory treatment [3]. Despite many scientific and social difficulties of drug discovery for tropical diseases, the major challenge remains the lack of economic prospects for investment in this field. Therefore, approaches aiming the rational design of compounds, such as SBDD, fragment screening, target-based drug discovery, and drug repurposing are of special interest. In this context, enzyme inhibitors offer unique opportunity for drug discovery. Nowadays about half of the known drug targets are enzymes and the rational to design new inhibitors has an established systematic approach [4]. In this review, we point to the advances on the field of SBDD targeting pathogen enzymatic pathways, including successful examples, major gaps on the field and perspectives for the future of drug design for tropical diseases.
Edited by Philip Cole and Andrea Mattevi
Antiviral drug targets https://doi.org/10.1016/j.sbi.2019.02.014 0959-440X/ã 2018 Elsevier Inc. All rights reserved.
Introduction Many infectious diseases are transmitted by arthropod vectors, being of special concern for countries situated at the tropical and subtropical regions. With about 40% of the world population living in risk areas, diseases such as dengue and malaria cause enormous health and economic impact on developing countries, especially in www.sciencedirect.com
Arbovirus, a contraction of ‘arthropod-borne virus’, is a term used to designate those viruses transmitted by insect vectors, usually ticks and mosquitos. Members of the families Flaviviridae, Peribunyaviridae, Phenuiviridae, and Togaviridae are known to cause human and animal diseases in a complex transmission cycle involving the virus, vertebrate host and the vector. Among the members of these families, the flaviviruses Yellow Fever (YFV), Dengue (DENV), Zika (ZIKV), and West Nile (WNV), as well as the togaviruses Chikungunya (CHIKV) and Mayaro (MAYV) are some of the emergent and reemergent arboviruses of public health importance in tropical and subtropical countries [5]. Dengue fever alone affects about 100 million people with clinical symptoms every year, with more than half of the Current Opinion in Structural Biology 2019, 59:65–72
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world population living in risk areas [1]. The constant resurgences of YFV in Africa and South America and the 2015–2016 epidemic outbreak of ZIKV in the Americas are examples of the great impact of flaviviruses in public health [6,7]. CHIKV outbreaks have been reported in Africa, Asia, Europe and America over the past 5 years, causing large epidemics with a higher incidence of more severe forms of the disease than previously reported [8]. Although there are vaccines to prevent YFV infections, specific treatments for any arbovirus-associated diseases are still not available [9]. The flavivirus genome is a positive-sense single-stranded RNA that encodes ten non-redundant proteins: three structural proteins that forms the viral capsid, membrane and envelope, and seven non-structural proteins (NS) that are responsible for the replication of the viral RNA inside the host cell [10]. The N-terminal domain of NS3 is a trypsin-like serine protease (NS3Pro) which requires the cofactor activity provided by the membrane-bound NS2B to process the viral polyprotein into mature proteins [11]. NS3 C-terminal region forms the RNA helicase domain responsible for unwinding the double stranded RNA before elongation [12]. The RNA elongation is performed by the C-terminal domain of NS5, which is an RNAdependent RNA polymerase (RdRp). The nascent positive RNA is capped at the NS5 N-terminal methyltransferase domain. All those enzymatic functions are essential for the viral replication cycle and, therefore, natural targets for antiviral drug discovery [13]. Over the last few years, the development of new inhibitors for flaviviruses targets has significantly grown due to the abundance of structural information for NS3 and NS5, together with well-established enzymatic and cell assays. The most studied protease inhibitors are peptide-derived molecules, such as peptide aldehydes and peptide boronic acids that shown a low micromolar to nanomolar inhibition of DENV serotype 2 (DENV-2) [14], WNV [15,16], and ZIKV NS3Pro [17]. However, potent peptide inhibitors of NS2B–NS3 protease detected in enzymatic assays often fail in cell-based assays, probably due to the low permeability of peptide derived compounds [18]. Yet, considering the success of HIV/AIDS protease inhibitor cocktails, this target remains a promising candidate for the development of broad-spectrum antiviral molecules [3]. The NS3 helicase structures have been reported for many flaviviruses, including DENV-2 [19], YFV [20] and ZIKV [21]. Besides the helicase activity, this enzyme also has an RNA50 triphosphatase activity (RTPase) essential to ensure its function in viral replication. A few inhibitors of the DENV NS3 helicase have been studied, like the benzoxazole analogue ST-610 [22] and a pyrrolone derivative [23]. Nevertheless, the lack of specificity of the RNA and NTP binding sites presumably leads to a significant toxicity as compounds targeting these sites might also bind Current Opinion in Structural Biology 2019, 59:65–72
to similar cellular proteins with helicase/NTPase activities. Still, most of the ligand-target structural information available is restricted to the ATP binding site of NS3 helicase (Figure 1). New structural-based techniques aiming to identify ligands for the RNA cleft are urgently required. The methyltransferase domain of NS5 is a small and globular protein, with a GTP and a S-adenosyl methionine binding pocket [24]. Many analogs of these molecules, such as Sinefugin, exhibited high affinity for the enzyme, but low effect on infected cells due to permeability issues [24]. Although structural information of many ligands is available and SBDD is possible, the accumulated failures in transposing the in vitro to in situ results turned the methyltransferase into a less interesting target for drug discovery [9,24]. In contrast, the RdRp domain of NS5 is probably the most interesting target for SBDD in flaviviruses. With a long and conserved cleft for accommodating the RNA during elongation, the structural data reveal many putative binding sites for SBDD [25]. The first non-nucleoside candidate reported to interact directly with the NS5 was Compound 27, an acyl sulfonamide analogue with potential antiviral activity against all four dengue serotypes, but not much is known about its subsequent development [25]. Other non-nucleoside inhibitors, such as ivermectin or fenretinide, are described to block the NS5 IMP-a/b1 interaction pathway in cells, but still no clinical trials were performed to test their efficacy [24,26]. Perhaps the most important lesson for designing inhibitors for flavivirus comes from another branch of this virus phylogeny. The Hepatitis C virus (HCV) is a member of the Flaviviridae family, in the genus Hepacivirus, and many genomic and structural similarities are shared among the groups. In 2013 a nucleotide analog, denominated sofosbuvir (PSI-7977), was approved for combination treatment of the three serotypes of HCV [27]. This prodrug is a phosphoramidate that is metabolized into a nucleotide analog that blocks RNA elongation in HCV NS5RdRp [27]. The oral administration of sofosbuvir in combination with a NS5A inhibitor, such as ledipasvir, has demonstrated impressive results in the treatment of chronicle HCV infections [28]. The mechanism in which the drug inhibits the HCV Genotype 2A RdRp via blocking RNA elongation was depicted with the ternary crystal structure of the complex (Figure 1) [29]. The methyluridine portion of the drug stacks to the RNA via Watson–Crick pairing, while the fluoride bounds to Asn291, selectively inhibiting the viral replication at the nanomolar range [29,30]. Despite the low sequence identity between the RdRp of HCV and other flavivirus (10–18%), a few residues involved in PSI-7977 stabilization are mostly conserved (Figure 1). Yet, stereochemistry and charge distribution of the relative region are remarkably different, meaning that further optimization may be required for the application of this molecule. Still, recent reports indicate that PSI-7977 might exhibit strong antiviral www.sciencedirect.com
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Figure 1
Lys141
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(a) Crystal structures of NS5 RdRp-DNA-Sofosbuvir complex from HCV showing details of (a) the mechanism in which the drug blocks RNA elongation by competing with nucleotides and (b) the relative position of Sofosbuvir in Zika virus crystal structure. For both, nucleosides are colored in green and surface is colored according to calculated charge from 5 kV (red) to 5 kV (blue). (c) Superposition of all available NS3 helicases from flavivirus (blue) with ligands show as red spheres, showing the clear lack of structural information the RNA cleft. Subdomains (SD) are depicted with orange dashes, ATP site with purple dashes and RNA cleft is demarked with arrow. (d) Structure details of Dengue virus RdRp in complex with compound 27.
activity against ZIKV, suggesting a broad-spectrum activity for nucleoside analogs [31,32]. The Alphavirus genome consists of a single stranded and positive sense RNA with two open read frames (ORF). The 30 ORF encodes five structural proteins: capsid C, envelope glycoproteins E1, E2, and E3, and the protein 6K. The 50 ORF encodes the four non-structural proteins NSP1, NSP2, NSP3, and NSP4, which the structure association results in the viral replication complex [33]. Apart from the NSP2 protease domain and NSP3 macro domain, wherein structures are currently available at the Protein Data Bank, the alphavirus non-structural proteins NSP1, NSP2 helicase domain and NSP4 have not been elucidated yet. Despite of that, NSP1 and NSP4 are considered attractive molecular targets for alphaviruses, as well as NSP2, and they have been explored by integrating in silico screen strategies and cell-based assays [34,35]. The NSP1 has guanine-7-methyltransferase (MTase) and guanylyltransferase (GTase) activities, directing the methylation and capping of the viral genomic RNA, essential to www.sciencedirect.com
prevent its exposure to host cell exonucleases. Since the non-conventional alphavirus RNA capping mechanism differs from the host cell mechanism by the sequence of reactions, NSP1 has been considered an attractive target to develop selective antiviral drugs [36]. Studies suggests that quinine, an antimalarial drug that reduces CHIKV replication in vitro, affects the NSP1 function [37]. More recently, in vitro screening against CHIKV strains associated with enzymatic assays for Venezuelan equine encephalitis virus (VEEV) suggested a novel class of triazolopyrimidinones as potent CHIKV replication inhibitors. Specifically targeting the guanylylation step catalyzed by NSP1, the lead compound MADTP-372 prevented the cytopathic effect (CPE) induced by CHIKV with an EC50 value of 2.6 mM. These results suggest that MADTP compounds are a promising antiviral therapy for CHIKV infections, although preclinical and clinical studies still need to be performed [38]. Alphavirus NSP2 is a multifunctional protein with a Cterminal protease activity necessary to cleave the Current Opinion in Structural Biology 2019, 59:65–72
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polyprotein NSP1234 into the four mature NSPs. Regardless of the low sequence identity, the NSP2 fold is highly conserved between species and its structure was elucidated for VEEV, CHIKV, and Sindbis virus. Using a NSP2 CHIKV comparative model, approximately 5 million compounds were screened in silico, a hit compound was selected and structural analogs were evaluated in vitro using a CHIKV cell-based assay. Compound 25, a acrylohydrazide derivative, the most potent molecule was shown to inhibit the CHIKV induced CPE with an EC50 of 3.2 mM. [39] Further studies have stablished structure–activity relationships, proposed a binding model for structural analogs in the proteolytic site of CHIKV NSP2 and identified novel and selective CHIKV induced CPE inhibitors at the low micromolar range. Additional structural modifications are ongoing to improve the antiviral activity of those inhibitors [40]. The NSP4 is an RNA-dependent RNA polymerase (RdRp) with well conserved motifs between positivesense RNA viruses. On the basis of that, a CHIKV NSP4 structure was built by comparative modelling
and explored by docking, aiming to investigate the binding mode of sofosbuvir [41]. The interactions observed between sofosbuvir and CHIKV NSP4 are similar to those observed for the natural substrate uridine triphosphate (UTP). Moreover, in vitro assays have shown that sofosbuvir inhibits the CHIKV replication in Huh-7 hepatoma cells and astrocytes derived from induced pluripotent stem cells. Furthermore, it has also been observed a systemic protective effect of sofosbuvir in vivo, which prevents the inflammatory cell increase caused by CHIKV. Altogether, those findings highlight the possibility that genetically distinct viruses such as Togaviridae and Flaviridae members could also be susceptible to sofosbuvir and propose clinical investigations on the use of this drug as a treatment for CHIKV infection [41].
Antiparasitic drug targets The malaria disease is caused by Plasmodium spp. protozoan and remains a significant global health challenge, killing more than 400 000 people each year, the majority being children under 5 years old living in sub-Saharan Africa [2]. The efficacy of antimalarial drugs has
Figure 2
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(a) Structure of Plasmodium falciparum dihydroorotate dehydrogenase. (b) Binding details of the Plasmodium falciparum dihydroorotate dehydrogenase bound to DSM265 (c) Crystal structure of Pfdhfr-ts. (d) Binding details of CYP51 from Trypanosoma cruzi in complex with posaconazole. Current Opinion in Structural Biology 2019, 59:65–72
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Antiviral and antiparasitic enzyme drug targets de Godoy et al. 69
Figure 3
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Key compounds discussed in this manuscript.
decreased due to the ongoing resistance of the parasite against clinically available drugs, including the current artemisinin-based combination therapy, which reinforces the extremely urgent need for new antimalarials [2]. www.sciencedirect.com
Over the last decade, many potential antimalarial drugs have been identified by phenotypic assays and subsequently demonstrated to target enzymes involved in hemoglobin hydrolysis, protein synthesis, pyrimidine Current Opinion in Structural Biology 2019, 59:65–72
70 Catalysis and regulation
biosynthesis, post-translational modifications and mitochondrial metabolism. A recent study tracked multiple genes that are potentially involved in the resistance acquisition by the parasite, including two new transporters and several enzymes as dihydrofolate reductase– thymidylate synthase (Pfdhfr-ts), farnesyltransferase, dipeptidyl aminopeptidase 1, and an aminophospholipid-transporting P-type ATPase [42]. Except for the Pfdhfr-ts (Figure 2), the high-resolution structures of these enzymes are still unknown, preventing the complete understanding of the underpinning molecular mechanisms and further structure-based optimization. A successful example derived from structure-based studies is the DSM265 compound, a triazolopyrimidine class inhibitor, which is currently in phase II clinical trials conducted in patients infected with P. vivax and P. falciparum [43]. Its target, the enzyme Dihydroorotate Dehydrogenase (DHODH), is a flavin (FMN)dependent mitochondrial enzyme, involved in a key step of the nucleic acid synthesis pathway (Figure 2). Unlike humans, Plasmodium relies entirely on the de novo pathway to acquire pyrimidines for DNA and RNA synthesis. DHODH is composed of a core b/a-barrel with an Nterminal a-helix that interacts with the mitochondrial membrane allowing the binding of Coenzyme Q10 (CoQ). The DSM265 inhibitor-binding pocket sits between FMN and the N-terminal a-helix, likely to be the binding-site for CoQ (Figure 2). This pocket is primarily hydrophobic and only two hydrogen bonds are formed between DSM265 and the protein via Arg-265 and His-185. The amino acid composition of the binding-site is highly variable between the Plasmodium and human enzymes and this property is thought to underlie the strong selectivity for the parasite over human DHODH [44]. Another compound currently in phase II clinical trial is MMV390048, a 2-aminopyridine derivative, which targets the phosphatidylinositol 4-kinase (PI4K), an enzyme that phosphorylates lipids to regulate intracellular signaling and trafficking in Plasmodium spp. The inhibitor is efficacious in a single dose and acts in all life cycle stages of the parasite [45]. Phase I compounds include P218, an inhibitor of dihydrofolate reductase; SJ733, a PfATP4 inhibitor; and M5717, an inhibitor of translation elongation factor 2, essential for protein synthesis [46,47]. Chagas disease, caused by the kinetoplastid parasite Trypanosoma cruzi, affects 6–7 million people worldwide, killing about 10 000 people annually. The disease is endemic in Latin America and the current treatment is limited to two rather old and suboptimal nitroheterocyclic drugs, benznidazole and nifurtimox. Significant efforts have been made to repurpose antifungal azole drugs targeting sterol biosynthesis, like CYP51, one of the most studied molecules, to treat Chagas disease Current Opinion in Structural Biology 2019, 59:65–72
(Figure 2). However, in controlled clinical trials, two antifungals, posaconazole (Noxafil, Merck) and ravuconazole (E1224, Eisai, Tokyo) showed inferior potency and side effects in comparison with the current treatment with benznidazole [48]. A similar case happens with visceral leishmaniasis, caused by the Leishmania spp. The disease causes about 40 000 deaths per year, and the available treatments are far from ideal with high cost, toxicity, and without clear mechanism of actions determined. A new promising lead molecule, GSK3186899/DDD853651, a pyrazolopyrimidine derivative, apparently acts by inhibiting the parasite cdc-2 related kinase 12, and showed great efficiency in mouse models. The efficiency of these compounds in clinical trials is yet to be tested [49]. Another bicyclic nitroimidazole molecule (DNDI-0690) showing excellent activity in vitro against both visceral and cutaneous leishhave progressed toward preclinical maniasis, development and a decision to progress to Phase I single ascending dose in healthy volunteers was agreed in 2018 [50].
Conclusion Despite many advances in the drug discovery field, most tropical diseases remain without a satisfactory treatment and our understanding of the interactions between host and pathogen is quite limited. Here, we highlighted some potential new inhibitors of enzymatic pathways of viral and parasitic pathogens of public health importance (Figure 3). There are still major gaps in this field, including the lack of structural information and mechanism of actions validated. Nevertheless, the accumulated knowledge from phenotypic screens in combination with biophysical and computational methods and the development of new sequencing and genome modification techniques allowed the discovery of many new putative targets for drug discovery.
Conflict of interest statement Nothing declared.
Acknowledgements The authors would like to thank the funding from the Sao Paulo Research Foundation – FAPESP (CEPID grant # 2013/07600-3 and post-doctoral grants 2015/18192-9, 2016/19712-9, 2018/05130-3), The National Council for Scientific and Technological Development (grants 405330/2016-2, 307132/2016-1, 151058/2018-0) and the Instituto Serrapilheira (#grant Serra1708-16250).
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