Citation: Matthew Brecher, Jing Zhang, Hongmin Li. The Flavivirus Protease As a Target for Drug Discovery .VIROLOGICA SINICA, 2013, 28(6) : 326-336.

The Flavivirus Protease As a Target for Drug Discovery

  • Corresponding author: Hongmin Li,
  • Received Date: 08 October 2013
    Accepted Date: 01 November 2013
    Published Date: 14 November 2013
    Available online: 01 December 2013
  • 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.

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    The Flavivirus Protease As a Target for Drug Discovery

      Corresponding author: Hongmin Li,
    • 1. Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany NY 12208, USA
    • 2. Department of Biomedical Sciences, School of Public Health, State University of New York, Empire State Plaza, PO Box 509, Albany, New York 12201-0509, USA

    Abstract: 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.

    • 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 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).

    • 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.

    • 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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