Many flaviviruses are significant human pathogens causing considerable disease burdens, including encephalitis and hemorrhagic fever, in the regions in which they are endemic. A paucity of treatments for flaviviral infections has driven interest in drug development targeting proteins essential to flavivirus replication, such as the viral protease. During viral replication, the flavivirus genome is translated as a single polyprotein precursor, which must be cleaved into individual proteins by a complex of the viral protease, NS3, and its cofactor, NS2B. Because this cleavage is an obligate step of the viral life-cycle, the flavivirus protease is an attractive target for antiviral drug development. In this review, we will survey recent drug development studies targeting the NS3 active site, as well as studies targeting an NS2B/NS3 interaction site determined from flavivirus protease crystal structures.
Citation: Matthew Brecher, Jing Zhang, Hongmin Li. The Flavivirus Protease As a Target for Drug Discovery[J]. VIROLOGICA SINICA, 2013, 28 (6): 326-336 https://doi.org/10.1007/s12250-013-3390-x
Received: 8 October, 2013; Accepted: 1 November 2013; Published: 14 November 2013
Copyright: © Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg 2013
Data Availability: All relevant data are within the paper and its Supporting Information files.
Corresponding author: Hongmin, Phone: +1-518-486-9154, Fax: +1-518-408-2190, E-mail: firstname.lastname@example.org.
Flaviviruses belong to the viral family Flaviviridae that include about 70 viruses (Brinton M A, 1981; Brinton M A, 2002; Westaway E G, et al., 1985). Many flaviviruses are significant human pathogens. Dengue virus (DENV) serotypes 1-4, Yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis complex virus (TBEV) are categorized as global emerging pathogens and are NIAID Priority Pathogens as well ( Burke DS, et al., 2001). Flaviviruses cause significant human disease, some of which are fatal such as dengue hemorrhagic syndromes and various encephalitides (Asnis D S, et al., 2001; Asnis D S, et al., 2000; Kramer L D, et al., 2001; Shi P Y, et al., 2002; Shi P Y, et al., 2002; Shi P Y, et al., 2001).
The World Health Organization has estimated annual human cases of 50, 000 for JE (WHO, 2009), 200, 000 for YF (WHO, 2009), and more than 50 million for Dengue fever (WHO, 2009). WNV is now the leading cause of arboviral encephalitis in the US, leading to more than a thousand human deaths (CDC, 2010; USGS, 2010). Morbidity and mortality rates are waning for WNV in the US, but are expected to increase for DENV. Currently, approximately 2.5 billion people are at risk of DENV infection, with an estimated 500, 000 cases in the form of life-threatening disease such as dengue hemorrhagic fever and dengue shock syndrome (WHO, 2009). However, vaccines for humans currently are available only for YFV, JEV, and TBEV ( Burke DS, et al., 2001); and more importantly no clinically approved antiviral therapy is available for treatment of flavivirus infection. Therefore, it is a public health priority to develop antiviral agents for post-infection treatment (Kramer L D, et al., 2007).
This article will review recent advances in flavivirus drug development targeting the essential viral protease.
The flaviviral genome structure
The flavivirus genome RNA, approximately 11 kb in length, is single-stranded and of positive (i.e., mRNA-sense) polarity. The viral genome consists of a 5' untranslated region (UTR), a single long open reading frame (ORF), and a 3' UTR (Fig. 1) ( Rice C M, et al., 1985; Shi P Y, et al., 2001). A cap is present at the 5' end, followed by the conserved dinucleotide sequence 5'-AG-3' (Cleaves G R, et al., 1979 ). The 3' end of the genome terminates with 5'-CUOH-3' (Wengler G, 1981) rather than with a poly(A) tract. The single ORF of flavivirus encodes a polyprotein precursor of about 3, 430 amino acids (Fig. 1A). The polyprotein is co-and post-translationally processed by viral and cellular proteases into three structural proteins (capsid [C], premembrane [prM] or membrane [M], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) ( Chambers T J, et al., 1990). The structural proteins form the viral particle and are involved in viral fusion with host cells including monocytes, macrophages and dendritic cells (Li L, et al., 2008; Lindenbach B D, et al., 2007; Marianneau P, et al., 1999; Tassaneetrithep B, et al., 2003). Low pH in the endosomal compartment triggers fusion of the viral and host cell membrane, which leads to the release of the nucleocapsid and viral RNA into the cytoplasm. This process is mediated by the viral E protein which is able to switch among different oligomeric states: as a trimer of prM-E heterodimers in immature particles, as a dimer in mature virus, and as a trimer when fusing with a host cell (Bressanelli S, et al., 2004 ; Modis Y, et al., 2004). The virus prM glycoprotein can be cleaved by furin protease to release the N-terminal "pr" residues during maturation, leaving only the ectodomain and C-terminal transmembrane region of "M" in the virion. The pr peptide protects immature virions against premature fusion with the host membrane (Guirakhoo F, et al., 1992; Li L, et al., 2008; Zhang Y, et al., 2003).
The NS proteins participate in RNA replication, virion assembly, and evasion of innate immune responses (Lindenbach B D, et al., 2007). The majority of the flavivirus NS proteins are multifunctional. NS1 is a large glycoprotein which is required for negative strand RNA synthesis (Lindenbach B D, et al., 1997; Lindenbach B D, et al., 1999; Muylaert I R, et al., 1997). NS2A has been reported to function in the generation of virus-induced membranes during virus assembly and/or release of infectious flavivirus particles ( Kummerer B M, et al., 2002; Leung J Y, et al., 2008). NS2B is a required cofactor for the protease activity of NS3 (Arias C F, et al., 1993; Chambers T J, et al., 1991; Chambers T J, et al., 1993; Falgout B, et al., 1993). NS3 is a large multi-functional protein with the activities of a serine protease (with NS2B as a cofactor), a 5′-RNA triphosphatase (RTPase), a nucleoside triphosphatase (NTPase), and a helicase (Li H, et al., 1999; Warrener P, et al., 1993; Wengler G, 1991). NS4A is an integral membrane protein involved in membrane rearrangements required to form the viral replication complex ( Miller S, et al., 2007; Roosendaal J, et al., 2006 ). NS4B has been reported to inhibit the type I interferon response of host cells, and might modulate viral RNA synthesis (Grant D, et al., 2011; Munoz-Jordan J L, et al., 2005; Umareddy I, et al., 2006). NS5 is the largest flaviviral protein with multiple enzymatic activities, namely the RNA-dependent RNA polymerase (RdRp) (Ackermann M, et al., 2001; Guyatt K J, et al., 2001; Tan B H, et al., 1996), the N-7 guanine and 2'-O ribose methyltransferase (Dong H, et al., 2012; Egloff M P, et al., 2002; Koonin E V, 1993; Ray D, et al., 2006; Zhou Y, et al., 2007), and the RNA guanylyltransferase (GTase) (Issur M, et al., 2009). Several NS proteins such as NS2A, NS4A, NS4B, and NS5 are thought to interfere with host immune responses (Ashour J, et al., 2009 ; Best S M, et al., 2005; Daffis S, et al., 2010; Guo J, et al., 2005; Munoz-Jordan J L, et al., 2003; Munoz-Jordan J L, et al., 2005).
The NS3/NS2B protease
The NS3 protein (~618 amino acids (aa)) is the second largest protein encoded by flavivirus. The N-terminal 170 aa of NS3 displays protease activity, and a hydrophobic core of about 40 aa in length within NS2B provides an essential cofactor function ( T J, et al., 1991; Chambers T J, et al., 1990; Falgout B, et al., 1991).The NS3 protease belongs to the trypsin serine protease superfamily with a catalytic triad (e.g. His51-Asp75-Ser135 for the DENV NS3) (Bazan J F, et al., 1989). The NS2B/NS3 protease complex prefers a substrate with basic residues (Arg or Lys) at the P1 and P2 sites and a short side-chain amino acid (Gly, Ser, or Ala) at the P1′ site (Chambers T J, et al., 1990; Gouvea I E, et al., 2007). The central function of the NS2B/NS3 protease complex is to process the flavivirus polyprotein precursor. As shown in Fig. 1 , the peptide bonds between capsid, NS2A-NS2B, NS2B-NS3, NS3-NS4A and NS4B-NS5 are cleaved by the NS2B/NS3 protease complex, leading to the release of mature individual NS proteins.
The NS2B/NS3 protease complex is essential for the flavivirus replication and virion assembly, as evidenced by the lack of production of infectious virions in mutants carrying inactivating viral proteases (Chambers T J, et al., 1993).
Crystal structure of the NS3/NS2B protease complex
The development of protease inhibitor began with the determination of the three-dimensional (3D) structures of the flavivirus NS3 protease, the NS2B/NS3 protease complex, and the protease-inhibitor complexes (Aleshin A, et al., 2007; Assenberg R, et al., 2009; Chandramouli S, et al., 2010; Erbel P, et al., 2006; Hammamy M Z, et al., 2013; Luo D, et al., 2008; Luo D, et al., 2010; Luo D, et al., 2008; Noble C G, et al., 2012; Robin G, et al., 2009). Currently, fourteen crystal structures of the NS2B/NS3 protease complex are available for the flavivirus N S2B/NS3 protease complexes, including the apo structures of proteases of WNV, DENV-1, DENV-2, DENV-4, and Murray Valley encephalitis virus (MVEV), the structures of proteases of WNV and DENV3 in complex peptide substrate-based inhibitors, and the broad-spectrum serine protease inhibitor aprotinin-bound structures of proteases of WNV and DENV-3.
In general, the flavivirus NS3 proteases display a chymotrypsin-like fold (Erbel P, et al., 2006). In all these structures, a NS2B fragment composed of about 44-47 amino acids, which provides an essential cofactor function (Chambers T J, et al., 1991; Chambers T J, et al., 1990; Falgout B, et al., 1990), is associated with NS3. When no substrate or inhibitor is present, the N-terminal (residues 51-61 in DENV-2) but not the C-terminal portion of NS2B is bound to NS3 (Erbel P, et al., 2006) (Fig. 1A). The central portion of this N-terminal part forms a β-strand and is part of the β-barrel of NS3 (Erbel P, et al., 2006). Consistent with the important structural role of this part of NS2B, structural comparison indicates that the NS2B residues within the N-terminal portion display similar conformations in all structures, regardless of presence or absence of inhibitors (Fig. 1A). It has also been reported that the N-terminal portion of NS2B (aa 49-66 only) is sufficient to bind and stabilize the NS3 conformation (Luo D, et al, 2008; Luo D, et al., 2010), although such a complex lacks protease activity (Luo D, et al., 2008; Luo D, et al., 2010; Phong W Y, et al., 2011). Mutagenesis studies demonstrated that two NS2B regions are critical for the protease function (Chappell K J, et al., 2008; Niyomrattanakit P, et al., 2004; Phong W Y, et al., 2011; Radichev I, et al., 2008) (Fig. 1D). Region one corresponds to the N-terminal region mentioned above, whereas region two is referred to a C-terminal region composed of residues 74-86 of NS2B. Residues within region one show great sequence conservation, especially for several hydrophobic residues at positions 51, 53, 59, and 61 (in DENV-2 order), with Trp61 strictly conserved (Fig. 1D). Functional studies indicated that three of these residues are essential, and the remaining one is also important, for the protease function (Chappell K J, et al., 2008). Structure comparison indicated that these conserved hydrophobic residues bind deeply into several pockets of NS3 (Fig. 1A). In contrast, residues within region two display greater sequence variation than those within region one, which may contribute to their fine substrate specificities as region two is part of the protease active site (see below) (Fig. 1B, 1C). In addition, in contrast to the N-terminal region which shows similar conformations, the C-terminal portion (beyond aa 61) of NS2B displays significantly large conformational differences between inhibitor-bound and inhibitor-free structures, and even between inhibitor-free structures (Fig. 1A). These results suggest that the N-terminal portion, but not the C-terminal portion, of NS2B is essential for NS2B to bind and stabilize NS3.
The C-terminal portion of NS2B has an integral role in active site formation in WNV and DENV. Although the C-terminal portions of NS2B display significantly different conformation in various apo crystal structures, the C-terminal portions of bound structures show remarkable conformational similarity when the complex is bound either to substrate analogs or the protease inhibitor aprotinin (Fig. 1B). In the structure of inhibitor-bound form, the C-terminal portion of NS2B forms a β-hairpin and "wraps around" the NS3 core, closing the NS3 active site. Several residues within this region make direct interactions, including hydrogen bonds, with substrate analogs or aprotinin inhibitors. Unsurprisingly, results from mutagenesis studies have demonstrated the importance of this region in protease function (Chappell K J, et al., 2008; Niyomrattanakit P, et al., 2004), likely due to its structural role in formation of the protease active site. The active site of the flavivirus NS2B/NS3 protease complex is quite flat and hydrophilic (Fig. 1C) and requires several basic residues as substrates, potentially hampering the development of potent competitive inhibitors.
Inhibitors for the NS3/NS2B protease
Viral proteases are proven antiviral targets. Numerous inhibitors against the HIV protease have been successfully developed and used in treatment of AIDS (Menendez-Arias L, 2010). Two HCV protease inhibitors have been recently approved to treat chronic HCV infections by FDA (Lin C, et al., 2006; Lin K, et al., 2006; Sarrazin C, et al., 2007). The success of protease inhibitors in other viruses has put the flavivirus protease in the focus of development for anti-flavivirus therapy. Both high throughput screening (HTS) and structure-based drug design have been explored to identify inhibitors against flavivirus protease.
Leung et al. reported the first inhibition studies using a recombinant covalently-linked NS2B/NS3 protease complex of DENV2 (Leung D, et al., 2001). Of sixteen standard serine protease inhibitors tested, however, only aprotinin, a basic pancreatic trypsin inhibitor, was shown to inhibit the enzyme with nanomolar IC50 (Drug concentration required to reduce enzyme activity by 50%) (Leung D, et al., 2001; Mueller N H, et al., 2007). Aprotinin was found to bind the NS2B/NS3 proteases of all four serial types of DENV with high affinity (picomolar) (Li J, et al., 2005); the in vivo efficacy of aprotinin in reduction of flavivi rus has not been reported. Nevertheless, although aprotinin is a potent inhibitor for the flavivirus NS3 protease, severe safety issues prevent it from being used as a drug. Aprotinin is a small protein which inhibits trypsin and related proteolytic enzymes and has been administered by injection, under the trade name Trasylol (Bayer) as a medication to reduce bleeding during complex surgery, such as heart and liver surgery, before 2007. The drug was permanently withdrawn worldwide in 2008 after studies suggested that its use increased the risk of complications or death (Mangano D T, et al., 2006; Mangano D T, et al., 2007).
Besides standard serine protease inhibitors, several peptidic α-keto amide inhibitors were also explored (Leung D, et al., 2001). Two peptidic inhibitor candidates showed inhibition activity for the protease with low micromolar IC50. Several similar peptidic inhibitor candidates, including cyclopeptides (Gao Y, et al., 2010; Xu S, et al., 2012), were found to be active for the NS2B/NS3 protease complex of DENV2, WNV, and YFV with Ki (the absolute inhibition constant) as low as 43 nM (Chanprapaph S, et al., 2005; Knox J E, et al., 2006; Nall T A, et al., 2004; Nitsche C, et al., 2012; Schuller A, et al., 2011; Yin Z, et al., 2006; Yin Z, et al., 2006). Although the in vivo efficacy of these inhibitor candidates has not been verified, the highly charged nature of these peptidic inhibitors may indicate poor bioavailability. The following studies seemed to verify this notion. Shiryeav et al. reported that the D-arginine-based peptides are potent inhibitors for the WNV NS3 protease, with Ki as low as 1 nM in an in vitro biochemical protease assay (Shiryaev S, et al., 2006). However, in a cell-based virus reduction assay, the inhibitor only showed micromolar inhibitory activity against the WNV (Shiryaev S, et al., 2006). In another study, Stoermer et al. reported that a peptidic inhibitor candidate showed high potency (Ki = 9 nM) for the WNV protease (Stoermer M J, et al., 2008). The inhibitor, composed of cationic tripeptide (KKR) with a phenacetyl-cap at the N-terminus and an aldehyde at the C-terminus, is cell permeable and stable in serum, but displays a much reduced antiviral activity (EC50 (concentration required for 50% viral reduction)=1.6 μM) (Stoermer M J, et al., 2008). The poor activities of these peptide-based inhibitors in cell-based assays may be explained by the poor penetration of charged peptides across the cell membrane. Nevertheless, the low bioavailability of these substrate inhibitors could limit their potential as effective chemotherapeutics (Chappell K J, et al., 2008; Noble C G, et al., 2010).
In addition to the standard inhibitors based on substrates, attempts to use protein as inhibitor has been explored (Rothan H A, et al., 2012). Rothan et al. reported that retrocyclin-1 (RC-1) can inhibit the NS2B/NS3 protease activity in vitro with IC50 in micromolar range. However, it only moderately reduced the virus growth even at 150 µM concentration.
Nonsubstrate based inhibitors were also investigated, though only moderate inhibition activity (IC50 in low micromolar range) was observed (Cregar-Hernandez L, et al., 2011; Ganesh V K, et al., 2005; Jia F, et al., 2010; Kiat T S, et al., 2006). To explore more small molecular inhibitors for the protease, both in silico-based and protein-based HTS has been developed (Aravapalli S, et al., 2012; Deng J, et al., 2012; Ekonomiuk D, et al., 2009; Ekonomiuk D, et al., 2009; Ezgimen M, et al., 2012; Gao Y, et al., 2013; Johnston P A, et al., 2007; Knehans T, et al., 2011; Lai H, et al., 2013; Lai H, et al., 2013; Mueller N H, et al., 2008; Nitsche C, et al., 2011; Samanta S, et al., 2012; Steuer C, et al., 2011; Tiew K C, et al., 2012; Tomlinson S M, et al., 2012; Tomlinson S M, et al., 2009). Several small molecule inhibitors were identified possessing low micromolar or high nanomolar inhibition activities for the WNV and DENV proteases (Bodenreider C, et al., 2009; Cregar-Hernandez L, et al., 2011; Ekonomiuk D, et al., 2009; Johnston P A, et al., 2007; Knehans T, et al., 2011; Lai H, et al., 2013; Mueller N H, et al., 2008; Sidique S, et al., 2009; Tomlinson S M, et al., 2011; Tomlinson S M, et al., 2009; Yang C C, et al., 2011). Although some of these compounds are potent inhibitors (IC50 up to 0.105 μM) for the flavivirus NS3 protease, some of them show poor stability with half life of only 1-2 h in solution (Johnston P A, et al., 2007). In addition, the majority of these studies, except the three discussed below (Mueller N H, et al., 2008; Tomlinson S M, et al., 2009; Yang C C, et al., 2011), did not use cell-based assays to evaluate the antiviral efficacy of identified compounds. In two studies (Mueller N H, et al., 2008; Tomlinson S M, et al., 2009), several compounds were found to inhibit the growth of WNV and DENV with EC50 in the low micromolar range. Furthermore, Yang et al. showed that a compound could inhibit the DENV NS3 protease with IC50 of 15 μM (Yang C C, et al., 2011). Encouragingly, this compound appeared much more potent in a replicon-based antiviral assay (EC50 of 0.17 M) than in the enzyme-based protease assay, possibly due to additional cellular targets.
All current approaches to identify inhibitors for the NS3 protease focus on the protease active site. However, only limited success has been achieved. This could be because the active site of the flavivirus NS3 protease is quite flat and highly charged (Aleshin A, et al., 2007; Assenberg R, et al., 2009; Chandramouli S, et al., 2010; Erbel P, et al., 2006; Luo D, et al., 2008; Luo D, et al., 2010; Luo D, et al., 2008; Robin G, et al., 2009), which makes it difficult to find small-molecule inhibitors of the NS2B/NS3 protease. Therefore, alternative approaches should be considered. Notably, the flavivirus NS3 protease requires NS2B as a co-factor for function. Therefore, the NS2B-NS3 association sit e may be targeted for identification and development of compounds that inhibit flavivirus NS3 protease function by blocking NS2B-NS3 association. The crystal structures of the NS2B/NS3 complex (Aleshin A, et al., 2007; Assenberg R, et al., 2009; Chandramouli S, et al., 2010; Erbel P, et al., 2006; Luo D, et al., 2008; Luo D, et al., 2010) and ample data from functional studies (Chambers T J, et al., 2005; Chappell K J, et al., 2006; Chappell K J, et al., 2008; Niyomrattanakit P, et al., 2004; Radichev I, et al., 2008) provide solid bases for HT screening of compound libraries to identify allosteric inhibitors. Currently, this approach has not been extensively explored. Only two reports indicated that a non-competitive inhibitor was identified, through a protein-based HTS assay, to have high potency against the NS3 protease, although one of the compounds was very unstable in solution (Johnston P A, et al., 2007; Pambudi S, et al., 2013). Docking experiments suggested that the compound binds to a site on the NS3 surface that may interfere with the binding between NS3 and the cofactor NS2B (Johnston P A, et al., 2007; Pambudi S, et al., 2013). Although a crystal structure of the inhibitor-NS3 complex is required to confirm the mode of action of this type of inhibitor, in vitro virus inhibition studies indicated that the compound identifi ed by Pambudi et al. that targets the NS2B-NS3 interactions can efficiently inhibit all four serotypes of DENV with EC50 of 0.74-4.92 µM (Pambudi S, et al., 2013). This compound also showed moderate inhibition activity toward YFV, indicating a potentially broad antiviral spectrum. Mutagenesis studies further revealed that mutations of DENV4 and YFV residues that were predicted to interact with the inhibitor candidate affected the sensitivity of viruses to this compound (Pambudi S, et al., 2013). These results strongly support the hypothesis that the interaction between NS2B and NS3 is a valid therapeutic target for anti-DENV drugs and argue that greater effort should be put towards developing allosteric inhibitors targeting the NS2B-NS3 interactions.
Historically, the most straightforward approach to developing inhibitors of an enzyme target has been to screen for compounds that competitively bind the enzyme's active site and displace native substrate. The advantage of such an approach is that characterization of the properties of a particular enzyme's substrate is often a sufficient starting point for selecting compounds that mimic or exceed the substrate in its affinity for the enzyme. Unfortunately, this approach might be unlikely to yield effective compounds in the case of flavivirus NS2B/NS3 protease for three reasons: First, NS2B/NS3 has a flat and hydrophilic active site which decreases the likelihood that compounds can bind specifically with high affinity. Second, the NS2B/NS3 active site is similar enough to those of host serine proteases that toxic effects in the host are likely for many compounds, as has been observed in the case of aprotinin. Third, the active site preferentially binds positively charged moieties; this charge can have deleterious effects on compound bioavailability.
In addition, lessons should be learned from the development of active site inhibitors for the HCV protease. Although two HCV protease substrate-based inhibitors were developed, resistant mutations occurred quickly (Wyles D L, 2013). This is because the active site of the HCV protease is shallow and solvent exposed. The featureless property of the active site of the HCV protease implies that inhibitors would rely on relatively few interactions with the enzyme for tight binding, resulting in a low barrier to resistance and extensive cross-resistance (Romano K P, et al., 2010; Wegzyn C M, et al., 2012). It has been reported that as few as a single key mutation resulted in a significant loss of inhibition and cross-resistance (Romano K P, et al., 2010; Wyles D L, 2012; Wyles D L, 2013). Similar to that of the HCV protease, the active site of flavivirus NS2B/NS3 protease complex is also flat and featureless, in addition to the hydrophilic nature. Therefore, potential drug resistance should be taken into account, when development of active-site inhibitors for flavivirus protease complex is considered.
Fortunately, the solved crystal structures of flavivirus protease in both substrate bound and unbound states has yielded mechanistic insight into protease function. Details of the interaction of the NS2B cofactor, critical for enzyme function, with NS3 have suggested an allosteric approach to inhibition through disruption of NS2B/NS3 binding. Lead compounds developed by this approach are less likely to have the drawbacks observed with active site inhibitors, and are amenable to both computational and HTS screening methods. In the future, this "structure-guided" approach may suggest additional allosteric sites in flavivirus protease and has the potential to open broad avenues to drug discovery in other disease target proteins.
This research was partially supported by grants (AI094335) from the National Institute of Health and from the Wadsworth Center Scientific Interaction Group.
All authors carried out the work presented here. MB, JZ, and HML wrote the paper the paper. MB and HML defined, reviewed and edited the theme of this review.
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