The Antiviral Potential of Host Protease Inhibitors
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The replication of numerous pathogenic viruses depends on host proteases, which therefore emerged as potential antiviral drug targets. In some cases, e.g., for influenza viruses, their function during the viral propagation cycle is relatively well understood, where they cleave and activate viral surface glycoproteins. For other viruses, e.g., Ebola virus, the function of host proteases during replication is still not clear. Host proteases may also contribute to the pathogenicity of virus infection by activating proinflammatory cytokines. For some coronaviruses, human proteases can also serve in a nonproteolytical fashion simply as receptors for virus entry. However, blocking of such protein-protein contacts is challenging, because receptor surfaces are often flat and difficult to address with small molecules. In contrast, many proteases possess well-defined binding pockets. Therefore, they can be considered as well-druggable targets, especially, if they are extracellularly active. The number of their experimental crystal structures is steadily increasing, which is an important prerequisite for a rational structure-based inhibitor design using computational chemistry tools in combination with classical medicinal chemistry approaches. Moreover, host proteases can be considered as stable targets, and their inhibition should prevent rapid resistance developments, which is often observed when addressing viral proteins. Otherwise, the inhibition of host proteases can also affect normal physiological processes leading to a higher probability of side effects and a narrow therapeutic window. Therefore, they should be preferably used in combination therapies with additional antiviral drugs. This strategy should provide a stronger antiviral efficacy, allow to use lower drug doses, and minimize side effects. Despite numerous experimental findings on their antiviral activity, no small-molecule inhibitors of host proteases have been approved for the treatment of virus infections, so far.
KeywordsAntiviral drugs Protease inhibitors Host targets Serine proteases Structure-based drug design
Crimean-Congo hemorrhagic fever virus
Human airway trypsin-like peptidase
Human immunodeficiency virus
Highly pathogenic avian influenza virus
Human parainfluenza virus
Influenza A virus
Influenza B virus
Lysine binding site
Lymphocytic choriomeningitis virus
Low pathogenic avian influenza virus
Middle East respiratory syndrome coronavirus
Protease-activated receptor 2
Severe acute respiratory syndrome coronavirus
Trypsin-like serine protease
Type II transmembrane serine protease
At present, 588 human proteases are listed in the degradome database (Quesada et al. 2009), which can be further divided into five classes based on their catalytic mechanism. The majority belongs to the family of metallo (192 members), serine (184 enzymes), and cysteine proteases (164 members); in addition, 27 threonine and 21 aspartyl proteases are known, so far. Proteases are well-druggable targets and numerous small-molecule protease inhibitors have been approved in the past. The more than ten inhibitors of the angiotensin-converting enzyme (ACE) (Turk 2006), as well as the neprilysin (enkephalinase) inhibitor prodrugs sacubitril (Howell and Cameron 2016) and racecadotril (acetorphan) target Zn2+-dependent metalloproteases. The ACE blockers and sacubitril are suitable for long-term usage as antihypertensive drugs, whereas racecadotril is approved as an antidiarrheal drug. Despite limited use, treatment of high blood pressure is also possible with aliskiren, a small-molecule inhibitor of the aspartate protease renin (Wood et al. 2003). Numerous synthetic inhibitors of the trypsin-like serine proteases (TLSP) thrombin (argatroban, dabigatran etexilate, bivalirudin) and factor Xa (rivaroxaban, apixaban, edoxaban, betrixaban) can be used as anticoagulants (Straub et al. 2011). Except argatroban and bivalirudin, all of these clotting protease inhibitors are orally available and suited for long-term use in the prevention of stroke, e.g., in patients suffering from atrial fibrillation. Meanwhile, more than ten gliptins have been approved in various countries. The gliptins are a class of oral hypoglycemic drugs for the treatment of diabetes mellitus type 2 targeting the serine protease dipeptidyl peptidase 4, thereby reducing the degradation of incretin hormones leading to an enhanced insulin secretion (Scheen 2015). The proteasome inhibitors bortezomib (Adams 2004) and carfilzomib (Kortuem and Stewart 2013) are used in patients with multiple myeloma and the first approved drugs targeting threonine proteases. Meanwhile, two additional proteasome inhibitors, ixazomib and oprozomib, obtained orphan drug status (Manasanch and Orlowski 2017). Besides addressing human proteases, numerous inhibitors of the aspartyl protease of HIV (Ghosh et al. 2016) and the NS3/4A serine protease of the hepatitis C virus (McCauley and Rudd 2016) are on the market. With few exceptions, most of these inhibitors are routinely used in combination with other drugs and not as single agents. Despite large efforts, no cysteine protease inhibitor has been approved, so far. One of the most advanced inhibitors of the papain-like bone-degrading protease cathepsin K is the nitrile derivative odanacatib, which was developed for the treatment of osteoporosis. Based on a clinical phase III trial, a high efficacy with increasing bone mineral density and reduced risk of fractures was initially reported, as well as a good safety profile (Chapurlat 2015). However, its further development was stopped at the end of 2016 due to a slightly increased risk of stroke (Mullard 2016). Other cathepsin K inhibitors, like the nitrile balicatib, failed in phase II due to complications with skin fibrosis (Brömme et al. 2016; Runger et al. 2012). Despite the lack of approved cysteine protease inhibitors, many other examples confirm the suitability of at least some proteases as excellent drug targets.
This should also apply to numerous host proteases, which are involved at various steps during the propagation cycle of certain viruses. The inhibition of host enzymes could be advantageous compared to the classical addressing of viral targets due to a low risk for rapid drug resistances. However, side effects may occur by targeting host enzymes, which are required for normal physiological processes. Moreover, most host proteases belong to families of structurally closely related enzymes, and therefore it might be challenging to address a single target without affecting other family members. In order to avoid side effects and a narrow therapeutic window, it is therefore advisable to develop such host protease inhibitors mainly for combination therapies, which should enable the use of lower drug doses.
In the following sections, numerous examples for the antiviral activity of inhibitors mainly addressing human serine proteases will be provided. In addition, structural aspects of the protease-inhibitor complexes will be discussed. Although first approved inhibitors of ACE and thrombin have been discovered long before the target structures have been determined, all subsequent successful developments in the field of protease inhibitors were strongly supported by the availability of crystal structures, which is an important prerequisite for a rational structure-based drug design.
11.2 Serine Proteases as Antiviral Targets
11.2.1 Trypsin-Like Serine Proteases
The majority of the human serine proteases belongs to the subfamily S1A, possessing a chymotrypsin-like folding pattern, and to the family S8 with the two subfamilies S8A and S8B exhibiting a subtilisin- or kexin-like folding (Rawlings et al. 2014). First studies on the limited proteolysis and essential maturation of viral glycoproteins by host proteases were performed on influenza viruses. It could be demonstrated that the cleavage of the hemagglutinin (HA) precursor could be blocked by the broad-spectrum serine protease inhibitor diisopropylfluorophosphate (DFP) (Klenk and Rott 1973). It was found that the poor infectivity of influenza A viruses (IAV) grown in cultures of chick embryo cells could be strongly increased after treatment with exogenous trypsin, which cleaves substrates after basic residues like arginine or lysine (Klenk et al. 1975). At the same time, comparable results were described for the trypsin-catalyzed activation of HA from influenza B viruses (Lazarowitz and Choppin 1975). Although the digestive protease trypsin is not found in the respiratory tract, these initial studies suggested that other trypsin-like airway proteases should be involved in HA maturation. For instance, plasmin efficiently activated the HA of the A/WSN virions but failed to cleave the influenza B HA (Lazarowitz and Choppin 1975). A detailed analysis of the substrate sequences revealed that HAs of human and other mammalian influenza viruses as well as HAs of low pathogenic avian influenza viruses (LPAIV) are cleaved after a single arginine residue before a constant P1′-P3′ Gly-Leu-Phe segment by trypsin-like serine proteases. In contrast, HAs of high pathogenic avian influenza viruses (HPAIV) are activated by furin-like proprotein convertases (PCs) at inserted multibasic sequences containing additional arginine or lysine residues in the adjacent non-primed positions (Garten and Klenk 2008). In addition to the HA activation of HPAIV, many other viruses depend on the correct cleavage of their surface glycoproteins by furin-like PCs (basic PCs) or by the neutral PC SKI-1 (Klenk and Garten 1994; Pasquato et al. 2013). Consequently, different inhibitor structures depending on the specific virus strains are required to target the appropriate activating protease.
The human genome encodes for approximately 70 different trypsin-like serine proteases, which cleave after a single basic residue, preferably after arginine in P1 position (Schechter and Berger 1967). Few of them, like matriptase or TMPSS13 (MSPL), strongly prefer substrates with additional basic residues in the non-primed region close to the cleavage site, e.g., in P4 and/or P3 position. The full-length enzymes can strongly differ in their molecular weights due to the presence of specific protein domains. However, all of them possess a relatively similar catalytic domain of approximately 225–230 amino acids with a strong structural homology to chymotrypsin of the subfamily S1A of serine proteases. This facilitates a common numbering of the residues within the catalytic domain with respect to chymotrypsin(ogen) used throughout this chapter. So far, crystal structures are available for ~25 different trypsin-like serine proteases. In most cases, the structures have only been determined for their catalytic domains, and their knowledge is normally sufficient for a rational structure-based design of active-site-directed inhibitors. The protease domain of the trypsin-like serine proteases consists of two six-stranded barrel domains, held together by several transdomain straps. The residues Ser195, His57, and Asp102 of the catalytic triad are located at the junction between these two barrels. All trypsin-like serine proteases contain a negatively charged aspartate residue at the bottom of their S1 pocket responsible for accepting substrates and inhibitors with basic P1 residues.
Data from virus-infected cell cultures suggested that different secreted trypsin-like serine proteases are involved in the HA activation of human IAV and LPAIV depending on the host and specific virus strain. A protease named tryptase Clara was isolated from Clara cells of rat airway epithelium (Kido et al. 1992). Furthermore, miniplasmin purified from rat lungs was described as potential HA activator (Murakami et al. 2001). Miniplasmin is a degraded version of plasmin, only comprising the kringle 5 and protease domain and lacking the N-terminal kringle domains 1–4 (Al-Horani and Desai 2014). However, plasmin cannot efficiently activate the HAs with monobasic cleavage sites found in the presently circulating influenza strains, because it prefers substrates with bulky P2 residues like Phe, Tyr, and Trp (Swedberg and Harris 2011). In embryonated chicken eggs, a blood clotting factor Xa-like protease was found to activate HA (Gotoh et al. 1990). Again, it seems unlikely that human factor Xa could be involved, which is a very specific protease with a strong preference for substrates with glycine in P2 position due to the presence of a bulky Tyr99 on top of its S2 binding pocket. This special structural feature limits the access of substrates with larger P2 side chains. So far, only prothrombin and eventually the protease-activated receptor 2 (PAR-2) are known as well-cleavable natural factor Xa substrates (Oe et al. 2016); both possess a glycine in P2 position. Moreover, in rats and mice, cellular ectopic trypsins due to severe influenza infections and cytokine storms were detected as HA-activating proteases (Kido et al. 2012; Pan et al. 2011; Wang et al. 2010). An unusual extrapancreatic trypsin expression was also found in some human tumors (Paju et al. 2001; Sorsa et al. 1997), raising speculations that this could also happen as a consequence of excessive inflammation during viral infections. Porcine tryptases are additional candidates (Chen et al. 2000; Sato et al. 2003), although human lung tryptase has failed to activate the HA of the 1918 IAV (Stevens et al. 2004). Among the 15 kallikrein-related peptidases (KLKs) (Goettig et al. 2010), 12 of them possess a trypsin-like substrate specificity, and a potential HA activation of seasonal IAV was observed for KLK5 and KLK12 (Hamilton and Whittaker 2013).
Additional candidates have been found among the type II transmembrane serine proteases (TTSPs) , which comprise 17 different enzymes (Antalis et al. 2011; Garten et al. 2015). Most of them show a restricted tissue-specific expression pattern; an exception is matriptase, which is ubiquitously found in epithelial layers of most tissues. In 2006, the TTSPs TMPRSS2 (epitheliasin) and human airway trypsin-like peptidase (HAT or TMPRSS11D) were identified as activating enzymes of monobasic HAs of seasonal IAV when overexpressed in MDCK cells (Böttcher-Friebertshäuser et al. 2010; Böttcher et al. 2006). A potential HA cleavage of the 1918 IAV was also described by TMPRSS4 (Bertram et al. 2010; Chaipan et al. 2009). The virus spread is eventually promoted by additional TTSPs, like DESC1 (Zmora et al. 2014) or hepsin, whereas no HA processing was found for TMPRSS3 and TMPRSS6 (also named as matriptase-2) (Bertram et al. 2010). In contrast to these TTSPs involved in the HA activation of LPAIV, a strong substrate preference for HAs from HPAIV with Lys as P4 residue has been identified for TMPRSS13 (MSPL) (Okumura et al. 2010). This is a striking difference to the substrate profile of the PC furin, which strongly prefers arginine in P4 position (Rockwell et al. 2002). Independent studies with knockout mice revealed that the HA of the seasonal H1N1 virions including the 2009 pandemic virus is dominantly activated by TMPRSS2 (Hatesuer et al. 2013; Sakai et al. 2014; Tarnow et al. 2014), whereas TMPRSS2 alone is not sufficient for the maturation of the HA from H3N2 subtypes. A recent study showed that only the combined TMPRSS2-/- and TMPRSS4-/- knockout mice reduced H3N2 spread and signs of infection in lung, suggesting that both proteases are involved (Kuhn et al. 2016).
220.127.116.11 Inhibitors for the Treatment of Human Influenza Virus Infections
In contrast to the trypsin complex, the Lys5 amino group makes only a water-bridged interaction to Asp189 at the bottom of the S1 pocket of matriptase but forms no direct contact to its carboxyl group. It also interacts with the side chain and carbonyl oxygens of the adjacent Ser190 and with a second water molecule (Fig. 11.3). Numerous analogues of SFTI-1 have been made. For instance, its conversion to the more simple truncated monocyclic analogue 3 provided a Ki value of 6.2 nM against matriptase (Fittler et al. 2013). The bicyclic analogue 4, containing a side-chain-to-tail cyclization, is even more potent with an inhibition constant of 2.6 nM (Fittler et al. 2014). However, despite this excellent potency, none of the SFTI analogues has been tested for antiviral activity.
Because of their high molecular weight aprotinin, HAI-1, HAI-2, or SFTI analogues should mainly inhibit extracellular HA processing, and it is rather unlikely that substantial amounts of these compounds can enter the cell and block the TTSPs in the secretory pathway. Despite this limitation, an aerosol formulation of aprotinin has been developed and approved in Russia for the treatment of mild-to-moderate influenza infections (Zhirnov et al. 2011). It was described that in case of mammalian IAV and LPAIV with monobasic HAs, progeny particles are assembled, which still contain some uncleaved HAs and therefore require further activation by extracellular enzymes sensitive to aprotinin. In contrast to these previous reports, more recent findings reveal that the monobasic HAs are processed in intracellular compartments without a further need for extracellular processing (Böttcher-Friebertshäuser et al. 2010, 2013). This suggests that small amounts of aprotinin can reach intracellular targets and achieve an antiviral efficacy (Zhirnov et al. 2011).
Substrate Analogue Inhibitors
Although an antiviral efficacy was demonstrated for ECA 5 and the S1 ligands 6–9, a stronger potency can be achieved with compounds also addressing adjacent binding sites. Trypsin-like host proteases possess relatively well-defined S2 and S3/S4 regions, often named proximal and distal binding pockets, respectively. Therefore, substrate analogue S3/S4-S2-segments were coupled with C-terminal decarboxylated P1 arginine mimetics or with stabilized non-cleavable P1-P1′ scaffolds, e.g., arginyl-ketone moieties or other warheads targeting the active-site serine.
The replacement of glycine by proline in P2 position leads to the relatively nonselective inhibitor BAPA (12), which possesses one- or two-digit nanomolar potencies against the clotting proteases thrombin, fXa, plasma kallikrein (PK), fibrinolytic plasmin, and the TTSPs HAT, TMPRSS2, and matriptase (Hellstern et al. 2007; Maiwald et al. 2016; Sielaff et al. 2011a). A significant antiviral effect was found in BAPA-treated cell cultures infected with numerous monobasic IAV strains (Böttcher-Friebertshäuser et al. 2010, 2012; Böttcher et al. 2009; Sielaff et al. 2011a). Many analogues of this inhibitor scaffold with different P2 and/or P3 residues have been tested but were less effective than BAPA (data not published). It seems that compounds with a broader target spectrum exhibit an advantageous antiviral profile. This tendency confirms the promising results obtained for the relatively nonselective inhibitor aprotinin. Otherwise, less selective inhibitors that also target numerous clotting proteases might suffer from a narrow safety profile due to potential bleeding complications after i.v. treatment. Such side effects might be reduced, when the compounds will be inhaled, as described for aprotinin. Notably, BAPA (12) was well tolerated in a bleomycin-induced fibrosis model in mice when it was applied as aerosol via a microsprayer (data not published).
In principle, it should be possible to replace the C-terminal 4-Amba residue in these substrate analogue inhibitors. Numerous alternative P1 groups are known from the field of thrombin and fXa inhibitors (Straub et al. 2011). Besides basic groups, many chloro-substituted aromatic P1 residues as used, for example, in inhibitor 13 have been developed (Sisay et al. 2010). The chlorine addresses a highly conserved Tyr228 side chain found at the back of the S1 pocket, whereas the aminomethyl group comes out of the S1 pocket and does not bind to Asp189. It provides an enhanced inhibitory potency against thrombin and fXa but leads to a reduced inhibition of HAT, matriptase, plasmin, and of several other trypsin-like serine proteases. However, only weak anti-influenza activity was found for compound 13 suggesting that the relevant HA-cleaving host proteases are not susceptible to such chloro-substituted aromatic P1 structures.
Covalent Substrate Analogue Inhibitors
Sulfonylated 3-Amidinophenylalanine Derivatives
Nonpeptidic Small-Molecule Inhibitors
Besides influenza viruses, other viral pathogens are also suitable targets for inhibitors of trypsin-like serine proteases (TLSPs). A few examples will be presented in the following paragraph.
18.104.22.168 TLSP Inhibitors for Treatment of Paramyxo- and Coronavirus Infections
Paramyxoviridae contain two surface glycoproteins, the receptor-binding protein (HN, H, G) and the fusion protein F. In contrast to the Orthomyxoviridae, where fusion of the viral membrane with host cell membrane occurs in the endosome, the activated F protein of many paramyxoviruses induces fusion of the virus envelope and the plasma membrane. The F0 precursor of human parainfluenza virus 1 (HPIV-1) is cleaved at a monobasic sequence typical for TLSPs (DNPQTR↓FFGAV) (Diederich and Maisner 2007). HPIV-1 causes respiratory infections, such as croup, especially among young children. In principle, some of the above-described inhibitors could also be suitable for the treatment of HPIV infections. With other paramyxoviruses, such as measles virus and mumps virus, F0 is activated by furin, whereas F0 of Nipah virus is cleaved by cathepsin L. Consequently, furin or cathepsin L inhibitors should be suitable for the treatment of these virus infections.
The spike (S) surface protein of respiratory coronaviruses (CoV) is also synthesized as inactive precursor protein that has to be activated by host proteases. The N-terminal S1 unit of the cleaved protein binds to receptors on host cells, and the C-terminal S2 unit enables the fusion of the viral membrane with host cell membranes. Both functions are essential for CoV propagation. Original studies suggested that the endosomal cysteine protease cathepsin L is solely required for spike activation and subsequent SARS-CoV infectivity (Simmons et al. 2005). Notably, a cathepsin dependency was also found for the enveloped Ebola virus of the Filoviridae family (Chandran et al. 2005) and Nipah virus (Diederich and Maisner 2007), as described above. Later, numerous groups have found that in the presence of cathepsin L inhibitors, the S-protein of SARS-CoV and MERS-CoV is activated by TMPRSS2 (Gierer et al. 2013; Matsuyama et al. 2010; Shulla et al. 2011; Zmora et al. 2014). Consequently, inhibition of SARS-CoV growth in Calu-3 airway epithelial cells was achieved by a combination treatment with the broad-spectrum serine protease inhibitor camostat (8) (Fig. 11.4) and the cathepsin inhibitor (23,25) trans-epoxysuccinyl-l-leucylamido-3-methyl-butane ethyl ester (EST) (Kawase et al. 2012). Based on these observations, it is suggested that SARS-CoV enters the host cells via two distinct pathways, one using TTSPs like TMPRSS2 and a second using the endosomal cathepsins L and/or B for spike activation. Interestingly, a recent study with a fresh clinical isolate of the human CoV 229E revealed a clear preference for host cell entry via TMPRSS2, whereas after 20 passages in HeLa cells the cathepsin L pathway became more important (Shirato et al. 2017). However, the cell culture virus showed a reduced ability for replication suggesting that the endosomal pathway is disadvantageous for HCoV-229E infection in humans. Based on these results, the authors suggested to target TMPRSS2 rather than endosomal cathepsins in CoV infections (Shirato et al. 2017). A similar tendency was previously found in an animal model of SARS-CoV infection, where viral spread and pathogenesis were only prevented by the TLSP inhibitor camostat and not by broad-spectrum vinylsulfone-type cysteine protease inhibitors targeting cathepsins L and B (Zhou et al. 2015). However, the authors argue that their new vinylsulfone inhibitors might be excellent lead structures for the development of inhibitors of Ebola virus entry.
11.2.2 Proprotein Convertases
Four PCs (furin, PC5B, PC7, and SKI-1/S1P) possess a transmembrane and C-terminal cytoplasmatic domain, which anchors them to cellular membranes. Furthermore, furin, PC5B , and SKI-1/S1P can be shed and released in a soluble form into the extracellular space. PC1 and PC2 are maintained in dense-core granules, whereas the remaining PC4, PC5A, PACE4, and PCSK9 are secreted. Furin and the related six PCs PC1, PC2, PC4, PC5, PACE4, and PC7 recognize multibasic cleavage sequences and, therefore, are also known as basic PCs or furin-like PCs. The catalytic domains of these enzymes show more than 50% identity (Thomas 2002). Due to the similar cleavage sites, a redundant behavior of these enzymes was found in overexpression experiments as well as in vitro studies. Despite overlapping consensus sequences and a high sequence homology, minor modifications in the recognition sequence as well as in their cellular localization lead to specific cleavages by the different PCs. In contrast, SKI-1/S1P cleaves after the consensus cleavage site (K/R)-X-(V/L/I)-Z↓, where Z is any amino acid except Val, Pro, Cys, Glu, or Asp and the spacer X is preferably a basic residue (Seidah 2013). After an autocatalytic cleavage at VFAQ↓S, PCSK9 forms a proteolytically inactive complex with its prosegment. So far, no other PCSK9 substrates are known.
Knockout of PCs in mice reveals their physiological significance especially during embryogenesis and contributes to the identification of specific PC substrates (Creemers and Khatib 2008; Seidah and Prat 2012; Taylor et al. 2003). Due to severe malformations, knockout of furin or SKI-1/S1P in mice leads to embryonic death, whereas PC5-deficient mice die at birth. For PACE4, a lethality of 25% was observed in knockout mice at embryonic day 14. In contrast, PC1- and PC2-deficient mice are viable but have several neuroendocrine peptide processing defects, and PC7 knockout mice show a loss of anxiety. The knockout of PC4 leads to infertility especially in male mice, whereas PCSK9 deficiency leads to lower plasma cholesterol levels.
The maturation of the PCs requires an autoproteolytic cleavage (Seidah and Prat 2012; Thomas 2002). In case of furin and furin-like PCs, two cleavages are needed to gain full enzymatic activity. After the removal of the signal peptide in the endoplasmic reticulum, a first cleavage leads to a conformational change of the enzyme, which is then the latent form. Enzymatic activity is obtained after a second cleavage in the prosegment, which leads to a release of the prosegment. The cleavages depend on the pH in the respective organelles. With exception of PC2 and SKI-1/S1P, the prosegments act as inhibitors of their respective enzyme. In case of SKI-1/S1P , three cleavages are required for full enzymatic activity. The subcellular localization of the PCs differs. Furin , PC5B , and PC7 have sorting signals in their cytosolic tails, which mediate recycling between the TGN and the cell surface. SKI-1/S1P shows also a broad distribution and is found in the ER, Golgi apparatus, endosomes, and lysosomes. In contrast, PC1 and PC2 are primarily found in dense-core vesicles of the secretory pathway, and PC4 is localized only in the plasma membrane of male and female germ cells. Furin, PC7, and SKI-1/S1P are ubiquitously distributed, and PC5 and PACE4 are widely distributed. In contrast, the expression of PC1 and PC2 is limited to neural and endocrine cells. PCSK9 can be found predominantly in the liver, intestine, and kidney.
The reported inhibitors against PCs can be categorized into various groups including macromolecular compounds, pure peptides, peptidomimetics, as well as nonpeptidic compounds, which will be described in the following sections.
22.214.171.124 Inhibitors of Basic Proprotein Convertases
A common approach for protease inhibitor development is the optimization of natural inhibitors by mutation of their inhibitory recognition loops. The α1-antitrypsin Portland (α1-PDX) is a bioengineered serpin-type inhibitor containing the furin-adapted sequence R355-I-P-R358 instead of A355-I-P-M358 in its inhibitory loop. α1-PDX inhibits furin with a Ki value of 0.6 nM in a slow tight-binding manner and is supposed to act as suicide substrate, yielding an inactive enzyme (Jean et al. 1998). It also inhibits PC1 (Ki = 260 nM) and PC5 (Ki = 2.3 nM) but has reduced potency against PC2, PC7, and PACE4 (Ki >1000 nM). Its expression in cells blocked the processing of HIV gp160 as well as measles virus fusion protein and, thus, inhibited virus spread (Anderson et al. 1993; Watanabe et al. 1995). Based on the reactive loop of α1-PDX, numerous mini-PDX peptides have been prepared. These acyclic- or disulfide-bridged cyclic 30-mers inhibit furin with IC50 values of 731 nM and 569 nM, respectively (Basak and Lotfipour 2005).
Turkey ovomucoid third domain (OMTKY3) belongs to the family of Kazal-type inhibitors and normally inactivates serine proteases of the S1A fold that prefer a neutral P1 residue. Exchange of A15-C-T-L18 to R15-C-L-R18 in its reactive site loop leads to a moderate furin inhibitor with an association constant Ka of 1.1 × 107 M−1 (Lu et al. 1993), which roughly corresponds to a reciprocal dissociation equilibrium constant of ~90 nM.
Like OMTKY3, inter-alpha-inhibitor protein (IαIp) is known to be a potent serine protease inhibitor, e.g., against trypsin, chymotrypsin, or acrosin. IαIp was first isolated from human plasma and is a multicomponent complex, consisting of two heavy and one light chain, called bikunin, which are linked via a chondroitin linker. Bikunin possesses two protease domains of the Kunitz-type that are likely to inhibit furin, because treatment with IαIp provided a significant protection against anthrax toxin in cell culture studies and in mice (Opal et al. 2005). Eglin C was originally isolated from the leech Hirudo medicinalis and belongs to the potato I inhibitor family. It inhibits several serine proteases, e.g., subtilisin, human leukocyte elastase, or cathepsin G. Insertion of a multibasic recognition site by mutation of P42-V-T-L45 to R42-V-K-R45 resulted in a strong furin inhibitor with a Ki value of 1.6 nM (Komiyama and Fuller 2000; Liu et al. 2004).
Additional furin inhibitors were designed by mutation of the homotetrameric glycoprotein α2-macroglobulin (α2-M), which is found in high concentrations in human blood. It is a potent broad-spectrum protease inhibitor with a unique inhibition mechanism. After protease mediated cleavage in the so-called bait region, the internal S-esters hydrolyze and trigger a conformational change of α2-M. The protease is enclosed by α2-M and sterically shielded from its substrates (Barrett and Starkey 1973). Replacement of its original G683-F-Y-E-S-D688 sequence by R683-S-K-R-S-L688 yielded a potent furin inhibitor, which blocked the processing of von Willebrand factor, TGF-β1, and HIV-1 gp160 (Van Rompaey et al. 1997).
The 45-kDa proteinase inhibitor 8 (PI8) was the first reported furin inhibitor, which is not a serpin reactive-site mutant. PI8 belongs to the group of ovalbumin-type serpins, containing two furin recognition sequences within its R336-N-S-R-C-S-R342 segment. The inhibition of furin by PI8 consists of two steps, starting with the rapid formation of a loose complex and followed by the slow isomerization to a stable complex. The overall Ki value for recombinant and soluble furin in vitro is 53.8 pM (Dahlen et al. 1998).
So far, only few endogenous PC inhibitors are known. A prominent role plays autoinhibition by prodomains (Zhong et al. 1999). Furin-like PCs are synthesized as zymogen and are activated by autocatalytic cleavage within the prodomain. The prosegment acts as an intramolecular chaperon, needed for the correct folding, regulation of enzymatic activity, and transport within the secretory pathway. A moderate Ki value of 156 nM was determined for the complete 83-mer prodomain of furin. Furthermore, this inhibitor reduced the proliferation, migration, and invasion of cancer cells (Basak et al. 2010). Several truncated derivatives have been synthesized. The most potent one, the 24-mer DYYHFWHRGVTKRSLSPHRPRHSR, inhibits furin with an inhibition constant of 0.9 μM. Moreover, some peptides derived from the prodomain of PC1 inhibit furin in the same range (Basak and Lazure 2003).
A combinatorial peptide library containing approximately 52 million hexapeptides was scanned to identify PC1 and PC2 inhibitors. For instance, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 inhibits PC1 and PC2 with Ki values of 3.2 and 360 nM, respectively, whereas it is only a moderate furin inhibitor with an inhibition constant of 1.4 μM. On the other hand, the unprotected analogue H-Leu-Leu-Arg-Val-Lys-Arg-OH has a stronger furin affinity (0.42 μM) but reduced potency against PC2 (3.4 μM) (Cameron et al. 2000).
Furthermore, inhibitor 35 (Fig. 11.16) containing a β-turn inducing enediynyl amino acid moiety was prepared. The amino and carboxyl groups of this unusual amino acid were coupled to peptide sequences around the cleavage sites within the prodomain of furin. This compound inhibits furin with a Ki value of 40 nM and blocks the cleavage of a fluorogenic peptide derived from the spike protein of human SARS coronavirus with an IC50 value of 193 nM (Basak et al. 2009).
Ki value (nM)
The inhibitory potency against furin could be further enhanced with poly-arginine derivatives, found by a positional scanning of combinatorial l- and d-hexapeptide libraries (Cameron et al. 2000). Hexa-d-arginine (39, Table 11.1) inhibits furin and PC5 with Ki values around 200 nM in the same range, whereas it is less active against PC1 and PC7. The elongated analogue nona-d-arginine-amide (D9R-amide, compound 40, Table 11.1) possesses a significantly improved inhibition constant of 1.3 nM. In contrast to the analogous l-peptide, which was cleaved by furin, the d-configured D9R-amide was found to be fully stable (Cameron et al. 2000; Kacprzak et al. 2004).
Another attempt to synthesize potent PC inhibitors is based on the monocyclic sunflower trypsin inhibitor SFTI-1 (Fig. 11.2) (Fittler et al. 2015). Therefore, the SFTI-1 backbone was used as a starting point for several modifications like the implementation of a furin cleavage motif and truncation of the inhibitor. The most potent compound (41) (Table 11.1) of this series inhibits furin in the low nanomolar range (Ki = 0.49 nM), whereas matriptase-1 is only poorly (Ki = 560 nM) and trypsin not affected.
Moreover, peptidomimetic compounds containing a multi-Leu motif have been described (Levesque et al. 2012). The aim of this modification was the discrimination between furin and PACE4. The most selective derivative of this series (42) inhibits PACE4 with a Ki value of 18 nM, whereas furin was 22-fold less affected. Further modification with polyethylene glycols of different length could improve the selectivity profile, whereas incorporation of 4-amidinobenzylamide or 2,3-dehydroagmatine as P1 residue resulted in less selective PACE4 inhibitors compared to furin (Kwiatkowska et al. 2016).
Furthermore, furin-inhibiting dicoumarol derivatives could be identified by HTS. Some of these compounds protected cells against furin-activated anthrax toxin and inhibited proMT1-MMP processing. Compound 49 noncompetitively binds to furin with an inhibition constant of 1 μM (Komiyama et al. 2009). Recently, a series of zinc and copper ion chelate complexes was described to inhibit furin in the micromolar range; the structure of the most suitable chelate ligand TTP (50) is shown in Fig. 11.19. The authors speculated that the active-site histidine might be coordinated by the zinc or copper ions. Interestingly, the solvated Zn2+ was less potent than its chelated form, whereby the free chelate ligands did not affect furin (Podsiadlo et al. 2004).
One approach to find new nonpeptidic furin inhibitors is the testing of compounds from natural sources. An example of this attempt is the screening of the chemical constituents of the medicinally used plant Andrographis paniculata. Derived from the major component andrographolide 51, several semisynthetic compounds have been tested. The most potent derivative is the andrographolide-trisuccinate pyridinium salt 52 (Fig. 11.19) with a Ki value of 2.6 μM (Basak et al. 1999).
126.96.36.199 SKI-1/S1P Inhibitors
Furthermore, enediynyl peptides were synthesized based on the amino acid sequence of cleavage sites of known substrate sequences like the LASV glycoprotein and the prodomain of SKI-1/S1P (Basak et al. 2015). The most potent compound (54, Fig. 11.20) inhibits SKI-1/S1P with a Ki value of 0.82 μM.
The concept of mutation and thereby optimization of natural serine protease inhibitors for a new target was also applied for the development of SKI-1/S1P inhibitors (Maisa et al. 2009; Pullikotil et al. 2004). Instead of the multibasic furin cleavage site, the SKI-1/S1P-specific motif (R/K)-X-X-(L/T)↓ (Seidah and Chretien 1999) was introduced. The overexpression of the α1-AT RRVL variant was found to inhibit both CCHFV (Crimean-Congo hemorrhagic fever virus) and LASV glycoprotein maturation. Blocking of arenavirus GPC processing had a strong antiviral effect in suppressing cell-to-cell spread and formation of viral particles. Micromolar concentrations of the wild-type prosegment of SKI-1/S1P were needed to inhibit the enzyme in vitro. A prosegment with the amino acid exchange R134E was the most potent mutant inhibiting the cleavage of CCHFV preGC (Pullikotil et al. 2004).
A set of nonpeptidic isocoumarinyl sulfone derivatives was tested against SKI-1/S1P. However, only one compound (57, Fig. 11.21) showed a weak inhibition of SKI-1/S1P (Ki = 255 μM) (Basak et al. 2015).
Several known serine protease inhibitors, among them AEBSF (4-(2-aminoethyl)-benzene sulfonylfluoride) and p-aminobenzamidine (Fig. 11.4) as well as inhibitors of furin or furin-related PCs like Dec-RVKR-CMK, were tested against SKI-1/S1P. A significant inhibition was only found for DCI (3,4-dichloroisocoumarin, compound 58, Fig. 11.21), which exhibited a slow irreversible binding mode with an apparent inhibition constant of 6.8 μM (Bodvard et al. 2007).
11.3 Miscellaneous Host Proteases as Antiviral Targets
Numerous cathepsin inhibitors have been prepared in the past. However, despite a few proof of concept studies, their deeper characterization and optimization as antivirals are limited, so far. They have been mainly tested in nonviral applications, e.g., for cancer therapy, osteoporosis, and rheumatoid arthritis or in neurodegenerative diseases (Siklos et al. 2015; Turk et al. 2012).
Zinc-dependent host metalloproteases from the MMP (matrix metalloproteases) or the ADAM (a disintegrin and metalloprotease) families might also be involved in the entry and fusion of certain viruses, as recently described for a neurovirulent murine CoV strain (Phillips et al. 2017). Moreover, a considerable amount of the EboV GP is shed by the metalloprotease TACE (ADAM17) (Dolnik et al. 2004). The soluble GP activates dendritic cells and macrophages and causes the release of pro- and anti-inflammatory cytokines and affects vascular permeability. The dysregulated inflammatory host response seems to contribute to the high virus pathogenicity (Escudero-Perez et al. 2014). These results suggest that inhibitors of metalloproteases may have antiviral activity. Although more than 50 clinical trials with metalloprotease inhibitors for the treatment of various cancers failed, their broad anti-inflammatory potential has aroused new interest (Vandenbroucke and Libert 2014).
11.4 Host Proteases as Receptors for Virus Entry
A few respiratory viruses use membrane-bound host proteases independent from their proteolytic activity as entry receptors. A surface region far away from the active site of the ubiquitously expressed serine protease dipeptidyl peptidase 4 (DPP4 , also called CD26) serves as human cellular receptor of MERS-CoV (Raj et al. 2013). Consequently, no antiviral effect could be observed after treatment of MERS-CoV-infected cells with active-site-directed DPP4 inhibitors. Moreover, the SARS-CoV and HCoV-NL63 use angiotensin-converting enzyme 2 (ACE2) (Li et al. 2003; Wu et al. 2009) and aminopeptidase N (Yeager et al. 1992) as human receptors. Both proteases do not show any sequence or structural similarity with DPP4 (Wang et al. 2013). The crystal structure of the receptor-binding domain of the MERS- and SARS-CoV S-protein in complex with DPP4 (Lu et al. 2013; Wang et al. 2013) and ACE2 (Li et al. 2005) has been determined, respectively. The complexes reveal typical protein-protein interactions (PPI). The receptor region on DPP4 and ACE2 are relatively flat missing deep binding pockets normally found in the active site of proteases. Although no examples are known so far, it should be possible to inhibit the entry of these virions by blocking the described PPIs with suitable ligands.
So far, only inhibitors addressing viral proteases have been approved for the treatment of certain virus infections. A huge arsenal of excellent inhibitors against host proteases has been developed in the past for treatment of chronic diseases, such as hypertension, diabetes, risk of thrombosis, inflammatory ailments, and cancer, but only few of them reached the clinic. Despite loss of patent protection, many of these failed inhibitors or their analogues could still be suitable for short-term treatment of acute life-threatening infectious diseases, without being hampered by side effects that might develop after long-term application. One of the most important prerequisites for successful drug development is the identification of a valid target. For some virus infections, the relevant host proteases have been identified, in other cases there are still uncertainties, and further basic research on target identification is needed. Since proteases usually belong to families of similar enzymes which substitute each other, a broad-spectrum inhibitor could be tolerable or even advantageous for the special treatment of infectious disease, although selective drugs are usually preferred for most applications to minimize side effects. Ideally, host protease inhibitors should be used in combination with additional drugs. This strategy should improve the antiviral efficacy and allow the use of reduced concentrations, thereby minimizing side effects. The development of effective and tolerable host protease inhibitors will hopefully expand the arsenal of antiviral drugs in the future.
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