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ACRYLATE AND VINYL SULFONE-BASED INHIBITORS OF VENEZUELAN EQUINE VIRUS CYSTEINE PROTEASE
Venezuelan equine encephalitis virus (VEEV) is a highly aerial infectious “New World” alphavirus of the Togaviridae family. This arthropod-borne (arbovirus) can be transmitted via mosquitos to birds, horses, and rodents and can cause severe encephalitis in humans. The virus is not only neuroinvasive but neurotropic; it replicates in the brain and lymphoid tissue. VEEV possess a nonstructural protein 2 (nsP2) cysteine protease that is vital to viral replication and is associated with the cytopathic effect (CPE) of the virus. Due to its role in the virus’s ability to replicate, it is a potential target for drug development and clinical intervention. Recently, a series of acrylate and vinyl sulfone-based inhibitors were identified as VEEV inhibitors, and we hypothesized that the molecular mechanism of the compounds is via inhibition of nsP2. The goal of this project is to test that hypothesis. To accomplish this, VEEV nsP2, and a FRET-based protein substrate (CFP-YFP) were recombinantly expressed in E. coli. The proteins were purified and used for FRET-based inhibition assays, as well as SDS-PAGE gel-based enzyme assays. The results showed that the compounds are indeed nsP2 inhibitors. The molecular interactions between the protease and the inhibitors are currently being investigated using X-ray crystallography. Future work will focus on structure-based optimization of the compounds’ antiviral activity.
TABLE OF CONTENTS
Alphaviruses: Old World and New World………………….1
Venezuelan Equine Encephalitis virus……………….4
Alphavirus Infection: Replication and Assembly………..6
nsP2 Functionality and Importance…………………8
Another antiviral Target……………………………..14
Current state of VEEV antiviral treatment……………15
List of Tables……………………………………..11
List of Figures…………………………………….12
Alphaviruses are a genus of arboviruses (arthropod-borne virus) from the Togaviridae family. These viruses are small, enveloped, icosahedral-shaped positive-sense RNA viruses approximately 65-70 nm in diameter, 12 Kb in length. The RNA genome translates to 4 nonstructural proteins used for viral replication and pathogenesis, and five structural proteins that make up the virion. Primarily segregated into two classes “Old World” and “New World” viruses contingent on the geographical location they were initially found (Byler, 2016). ‘Old World’ alphaviruses include Chikunga virus (CHIK), O’Nyong Nyong virus (ONNV), Ross River virus (RRV), Semiliki Forest virus (SFV), and Sindbis virus (SINV). Alphaviral infections with “Old World” viruses are typically related with fever, rash, and rheumatic diseases like polyarthralgia or polyarthritis. Despite this type of alphaviruses being chronic and incapacitating they are seldom fatal. ‘New World’ alphaviruses are Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and Venezuelan equine encephalitis virus (VEEV). Unlike ‘Old World’ alphavirus these viral infections are affiliated with possibly fatal encephalitis in humans and other mammals (Byler 2016). These types are considered to be “neglected” tropical illnesses for which there is no present antiviral treatments or vaccines accessible.
Chikungunya virus was first classified as a human pathogen in Tanzania in 1952. Its symptoms range from fever, skin rash, to debilitating arthralgia. CHIKV is primarily spread by the two mosquito species Aedes albopictus and Aedes aegypti. Several instances of chikungunya fever have been found in Africa, Asia, and more recently in Europe and the Western Hemisphere. In 2004, a large epidemic of CHIKV occurred in Kenya and spread throughout islands in the Indian Ocean. Between 2013-2014 an outbreak happened in the Caribbean region, and the virus has now been discovered in northern South Africa, Central America, and southeastern United States, causing more than 1 million CHIKV infections (Byler, 2016). O’Nyong-Nyong virus is closely associated with CHIKV. In 1959, ONNV had a considerable epidemic outbreak in East Africa, southern Uganda in 1996 and even arose in the Ivory Coast in 1996. Anopheline mosquitoes, Anopheles funestus and Anopheles gambia normally transmit ONNV, however, culicine mosquito, Mansonia uniformis have been found with the virus. The major arthropod vectors are Aedes vigilax, Aedes camptorhychs, and Culex annulifostris. The symptoms of O’Nyong-Nyong are similar to that of CHIKV infections, usually involving fever, macuopapular skin rash, pruritus, myalgia, and arthralgia (Byler, 2016). Ross River virus was first discovered in 1959 from Aedes vigilax mosquitoes caught along the Ross River in Townsville, north Queensland. In 1979, RRV swept into the South Pacific resulting in huge epidemics in New Caledonia, Fiji, Samoa and Cook Islands. Infections commonly cause rash, fever, myalgia, and arthralgia and sometimes arthritis (Byler, 2016). Semliki Forest virus, was first identified from Aedes abnormalis mosquitoes in the Semliki Forest of Uganda, but is usually found in sub-Saharan Africa and is predominantly distributed by Aedes africanus and Aedes aegypti mosquitoes. Generally SFV symptoms cause mild fever or can be asymptomatic, but virulent strains of the virus can cause deadly encephalitis in mice. In 1957, outbreak of SFV occurred in Central African Republic and several patients expressed fever, severe and chronic headache, and myalgia, and arthralgia (Byler, 2016). Sindbis virus was first found from Culex pipiens and Culex univittatus mosquitoes in the village of Sindbis, Egypt. Sindbis virus was the culprit of Pogosta disease in Finland, Ockelbo disease in Sweden, and Karelian fever in Russia and is spread by Culex spp. and Culiseta spp. mosquitoes. The viruses is prevalent in the Old World such as in Europe, South Africa, Australia, and China. It causes mild fever, itching rash, fatigue, myalgia and arthralgia (Byler, 2016).
Eastern equine encephalitis virus is a “New World” arbovirus, and is closely related to VEEV. It ranges from Eastern North America, through Central America, the Caribbean, and into South America as far south as Argentina. Rare in humans with only 82 confirmed cases of infections of EEEV in the U.S. between 2044-2013 mostly occurring within the Atlantic and Gulf States. Despite this, the disease is virulent with a mortality rate in people around 33% and significant brain damage in the majority of survivors. Enzootic transmission is between birds and ornithophilic mosquitoes and Culex spp. are possible bridge vectors for transmission to mammals in North America (Byler, 2016). Western equine encephalitis virus, similar to VEEV and EEEV, is common in western North America and South America. Like EEEV, the virus is preserved within a mosquito-bird transmission cycle with Culex tarsalis as the principal vector in western North America. Transmission to equine and humans is possible by “bridging” mosquito vectors, Ochlerotatus melanimon in California, Aedes dorsalis in Utah and New Mexico, and Aedes campestris in New Mexico. WEEV is asymptomatic, but the disease able to cause symptoms (fever, headache, nausea, malaise) in infants and the elderly with a mortality rate of around 4% (Byler, 2016).
Venezuelan equine encephalitis (VEEV) was originally isolated in 1993 from the brain of an infected animal in Venezuela. VEEV is confined to tropical and subtropical Americas, from Argentina to south Texas. It has at least 13 subtypes. In 1995, a major outbreak occurred in La Guajira, Columbia, of the serotype IC transmitted primarily by the Aedes taeniorhynchus mosquito (Byler, 2016). This particular outbreak was the cause of 75,000 human cases with 3000 neurological complications and 300 fatalities. People with serotype IC infection exhibited acute, self-limited fever, however convulsions were often observed in adolescents, and abortions and fetal death happened in pregnant women. Between 1969-1972 on the Guatemala-El Salvador border there was a major outbreak of VEEV of the serotype IAB, which spread northward through Mexico and into southern Texas, and caused about 50,000 equine deaths and 93 confirmed human deaths in Mexico (Byler, 2016). Infections with endemic VEEV strain ID has been the cause of at least 3% febrile disease in Iquitos, Peru. The ID serovar causes acute, undifferentiated fever, headache, malaise, and arthralgia (Byler, 2016)
Similar to WEEV and EEEV, VEEV is a zoonotic pathogen, transmitted between vector mosquitoes and vertebrate hosts. VEEV has two routes of transmission, enzootic and epizootic. The enzootic cycle is circulated among rodent and other vertebrates (rats, bats, opossums, etc.) as reservoirs and mosquitoes in subgenus Culex as primary vectors (Hu, 2016). The Epizootic cycle is transmitted by several mosquito vectors that feed on the susceptible amplification host (a host in which infectious agents multiply rapidly to high-levels), horses (Paredes, 2005). Essentially any mosquito can be found infected with VEEV during epizootics. However, Ochlerotatus taeniorhynchus is thought to be the primary vector accountable for transmission of VEEV during outbreaks, whereas Culex (Melanocion) species mosquitoes transmit enzootic strains of VEEV (Zacks, 2010). Equine are unusually susceptible to the virus, and fatality rates for horses are 20-80% (Hu, 2016). It is both neuroinvasive, being able to infect the nervous as well as neurotropic, having a tendency or affinity to infect the nervous system. The virus replicates in both lymphoid tissue and in the brain causing infections which are usually combated with innate and adaptive immunity (Hu, 2016). VEEV is extremely transmittable via aerosol route being the cause of numerous laboratory accidents (>150 cases without a corresponding puncture wound), and even has been established as a weapon of biological warfare in the U.S. and in the former Soviet Union. An uncommon characteristic of VEEV viral particles is that it is highly resilient to desiccation and can be stably lyophilized and aerosolized. If inhaled VEEV can pass into the brain through the olfactory neurons, supporting its use as a biological weapon (Hu, 2016).
The initiation of infection begins with a viral particle fusion with the host cell membrane or engaging with the receptor of a susceptible host cell (Leung, 2011). Primarily, entry into host cell is via receptor-mediated endocytosis, where clathrin-coated pits are formed, and the subsequent transport of early endosomes, where low-pH environments initiates fusion (Leung, 2011). Once inside the cell alphavirus capsid undergoes disassembly, releasing its genomic RNA into the cytoplasm of the host cell. Viral genomic RNA is then translated from two open-reading-frames to nonstructural and structural polyproteins (Leung, 2011). The nonstructural polyproteins that genomic RNA yields are either nsP123 or nsP1234. Typically, and primarily nsP123 is produced however, some alphavirus New and Old lack the opal codon, and only nsP1234 is translated (Shin, 2012).
At the start of infection P1234 is cleaved in cis between nsP3 and nsP4, yielding nsP123 and nsP4 by the carboxyl-terminal protease domain of nsP2. These two form an unstable preliminary replication complex able to synthesize negative-strand RNA (Leung, 2011). Next nsP2 cleaves P123 to nsP1 and P23 occurring in trans. The cleavage products nsP1, P23, and nsP4 form a replication complex within the virus-induced cytopathic vacuoles (CPV I) called spherules. The three polyprotein products play a role in negative-strand synthesis, as well as genomic RNA synthesis, not sub genomic RNA. After nsP2 cleaves the nonstructural polyprotein into its four mature parts: nsP1, nsP2, nsP3, and nsP4, negative strand RNA synthesis is terminated and the complex begins to synthesize positive-strand genomic and sub genomic RNA (Leung, 2011).
Translation of the positive sense subgenomic RNA creates a single structural polyprotein. It is cleaved into 5 structural proteins: the Capsid (C), two major envelope glycoproteins E1 and E2, and two small cleavage products E3 and 6K (Leung, 2011). Protein processing of the structural polyprotein happens cotranslationally, in which the capsid protein is the first to be cleaved from the polyprotein (Leung, 2011). This makes the C protein available to associate with newly created RNA. The C protein is able to recognize certain packaging signals in the 5’ portion of the genome, so that only full-length genomic RNA is packed into the nucleocapsid of the virion. The E3 protein serves as a signal sequence that directs the remaining of the poly protein to be inserted into the endoplasmic reticulum (ER), where it is processed by host signal peptidase (Leung, 2011). The 6K protein acts as a signal sequence for the downstream processing of the E1 protein. At the start of synthesis, PE2, E2 glycoprotein precursor, interaction with E1 glycoprotein forms heterodimers. These heterodimer complexes are then taken from the ER to the cell membrane via the Golgi apparatus. In a further state of transport, host cell furin-like protease cleaves PE2 in its lumental domain to generate mature E2 and E3 proteins (Leung, 2011). A conformational change is induced by this cleavage which in turn weakens the E1-E2 interaction in the spike heterodimers. This action primes the fusion peptide for activation upon exposure to low pH. The budding process is driven by the interactions between C protein and the cytoplasmic domain of the E2 protein, with E1-E2 heterodimers forming an envelope around the nucleocapsid like particles (Leung, 2011). Finally, during the release of the virions from the cell it attains its membrane lipid bilayer from the host cell membrane.
Nonstructural protein 2 has countless enzymatic activities and functional roles within an alphavirus specifically VEEV. Alphavirus replication and proliferation is reliant on the catalytic activity of the viral nsP2 protein, which as stated earlier cleaves the nsP1234 polyprotein replication complex into its mature parts. The N-terminal half possess a helicase domain that contains 7 signature motif of Superfamily 1 helicases (Bakar, 2018). It serves as an RNA triphosphatase that performs the initial viral RNA capping reactions, the N-terminal also acts as a nucleotide triphosphatase, driving, RNA helicase activity. The C-terminal region of nsP2 contains a papain-like cysteine protease, responsible for processing the viral non-structural polyprotein (Bakar, 2018). A cysteine protease also known as thiol proteases are involved in process such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins (Verma, 2016). Specifically, alphavirus nsP2 has also been suggested to be a virulence factor accountable for the transcriptional and translational shutoff of infected host cells. In addition, the inhibition of interferon mediated antiviral response is also a role of nsP2 which contributes to the controlling of translational machinery by viral factors (Bakar, 2018). Ultimately, making this protease, specifically nsP2 an excellent target as a drug target. NsP2 also cleaves substrates at a defined recognition sequence: Asp/Glu-Ala-Gly-Ala) or Glu-Ala-Gly-Cys in VEEV further supporting its significance as an antiviral target (Russo, 2006).
Despite nsP2 being an obvious and sensible target for drug application some other targets have been proposed. For example, during infection cytoplasmic vacuole formation is induced in the host cells (Bakar, 2018). These vacuoles have small membrane invaginations called spherules where the replication proteins nsP1 and 4, host factors as well as newly created viral RNA are contained (Bakar, 2018). These structures serve as localized areas to facilitate virus propagation, by allowing special isolation and propagation of RNA translation, replication and packaging of the viral genome. Spherules ultimately protect viral RCs and genomic RNA from host cell enzymes and prevent identification by antiviral double-stranded RNA sensors. NsP1 role during this process is that it recruits the other nsP into the spherules. Recent studies have demonstrated nsP1 involvement in the recruitment of the other nsPs into the spherules suggesting that its interaction with all the other nonstructural proteins is undeniably critical in keeping the RC intact and functional (Bakar, 2018). Ultimately, making nsP1 another likely target for drug development.
As of now there are no licensed vaccines or treatments for alphaviruses. However, there are two vaccines used under Investigational New Drug (IND) status for lab personnel protection (Hu, 2016). TC-83, is the the live-attenuated vaccine, which protects against both subcutaneous and aerosol exposure in animals, and ~82% of vaccinated humans develop immunity after vaccination (Hu, 2016). A derivative of TC-83 called C-84, a formalin-inactivated vaccine protected against subcutaneous infection, but was unsuccessful in protecting against aerosol exposure, which implies inadequate initiation of mucosal immunity (Hu, 2016). A couple of small molecular inhibitors targeting host or viral proteins have been discovered, however none have been identified to directly inhibit the nsP2 cysteine protease or have much effects on the WEEV or EEEV. Due to encephalitis being a disease of the brain most antivirals have limited permeability due to the blood brain barrier (Hu, 2016). The majority of alphaviruses are classified as being “neglected” tropical diseases affecting people living in impoverished conditions, and these people usually use naturopathy for their medicinal needs (Byler, 2016). Presently symptom-based treatment is utilized along with analgesics and non-steroidal anti-inflammatory agents.
Currently, infections with VEEV are diagnosed commonly by “direct detection, e.g., nucleic acid or virus isolation from acute-phase serum or spinal fluid or by serological assay, e.g.,detection of VEEV-specific IgM in the CSF using MAC-ELISA or monoclonal antibody-based antigen-capture ELISA (Zacks, 2010).” The PRNT or plaque reduction neutralization test, similar to the MAC-ELISA is useful in determining VEEV infection from other alphaviruses, however, cannot be utilized to determine the subtype (Zacks, 2010). Recently, a VEEV-specific blocking ELISA was described to be able to identify serotype specific antibodies against VEEV in sera of humans, equids or rodents. Nevertheless, no effective antiviral therapeutic exists for VEEV (Zacks, 2010).
The VEEV nsP2 protease was joined to thioredoxin (Trx) and hexa-histidine tag with a thrombin cleavage site in a pET32a vector. The His-tag was associated with thioredoxin. Escherichia coli were made competent to accept the Trx-VEEV-nsP2 plasmid. Luria Bertani (LB) media (4-8 L) containing 50µg/mL ampicillin (Amp) and 25µg/mL chloramphenicol was inoculated and grown to an OD600 of roughly 1.0 and induced with 0.5 isopropyl β-D-1-thiogalactopranoside (IPTG) overnight at 17° C. Cells were pelleted and lysed using 50 mM Tris pH 7.6, 500 mM NaCl, 35% BugBuster, 5% glycerol, 2mM β-mercaptoethanol (BME), 25 U of DNase, 0.3 mg/mL lysozyme and sonicated 10 times for 30 sec intervals in an ice bath. Lysates were centrifuged at 4600 rpm for 45 min, the supernatant was loaded onto a Ni-NTA nickel column equilibrated with 50 mM Tris pH 7.6, 500 mM NaCl, 2 mM BME, 5% glycerol. Flow through was collected. The column was then washed with same buffer containing 60 mM imidazole. Protein was eluted with the same buffer containing 300 mM imidazole. Dialysis was then conducted on the protein sample in 50 mM Tris pH 7.6, 250 mM NaCl mM ditheiothreitol (DTT), 1 mM ethylenediamine tetraacetic acid (EDTA), 5% glycerol, and then loaded onto a Hi-PrepTM SP XL 16/10 column equilibrated with 50 mM Tris pH 7.6, 5% glycerol and 5 mM DTT. A gradient of buffers with varying NaCl concentrations were used to further purify the VEEV nsP2 on the SP-column with 250 mM NaCL being the highest and 500 mM NaCL being the highest. A fraction collector was used to collect the samples and a SUREPageTM Gels were used determine the purity of the fractions. They were then concentrated using an Amicon Stirred Cell. The concentrated fractions were then loaded on to a HiLoad™ 16/600 Superdex™ 75 pg column to further purify. After purification the sample was concentrated again and thrombin was used to cut the fusion protein (thioredoxin and His-tag). The thioredoxin-His-tag free enzymes were used for FRET analysis. The buffer was exchanged to the appropriate assay buffer prior to all FRET assays using a PD-10 column. Protein concentration was determined from
Protein Construct Expression and Purification of FRET Protein Substrate
Escherichia coli were made competent to accept the pET15b plasmid (AmpicillinR) encoding cyan fluorescent protein (CFP), and nsP2 protease cleavage site motif, AG(A/C)↓(G/Y/A), and yellow fluorescent protein (YFP) in between the Ndel and Xhol cut sites was synthesized for each cleavage site. LB containing 50µg/mL ampicillin (Amp) and 25µg/mL chloramphenicol (4-8 L) was inoculated and grown to an OD600 of roughly 1.0 and induced with 0.3 mM IPTG overnight with shaking at 17℃. Cells were pelleted by centrifugation, lysed with lysis buffer (50mM Tris pH 7.6, 500 mM NaCL, 2µl Bug Buster, 2mM BME), and briefly sonicated for 1 minute in an ice bath. Lysates were clarified by centrifugation (4500 rpm for 30 minutes at 4℃ and loaded onto a nickel column equilibrated with 50mM Tris pH 7.6, 500 mM NaCL, 2 BME. The column was washed with the same buffer after loading, and with 10-20 column volumes of buffer containing 60 mM imidazole. The protein was eluted with the same buffer containing 300mM imidazole. The protein was dialyzed against 50 mM Tris pH 7.6, 150 mM NaCl overnight at 4℃. All substrates were produced at high yield (usually 60-80 mg per liter of media) and could be readily concentrated to 9.0-10.5 mg/mL. The substrates were used for continuous and discontinuous assays.
Continuous Gel-Based Assay
Cleavage of the YFP/CFP FRET substrate was monitored continuously at room temperature using excitation/emission wavelengths of 430/470 nm and 430/570 nm and Omega plate reader. The substrate was buffer-exchanged into .1M sodium phosphate buffer pH 6 using PD-10 columns. Enzyme concentrations ranging from 12.5-850 µM (7 different concentrations) were used in assays. Data was collected in triplicate (50 µL reaction volumes) in half-area black low binding surface 96-well plates from Corning, Inc. Plates were read for 10-120 minutes at 1-2 minute intervals. After the reads were completed the plates were sealed with film and allowed to digest overnight at room temperature and final emission ratios were read the next day. The fraction of substrate cleaved, f, was calculated from the emission ratios at each time point using the formula equation:
Discontinuous Gel-Based Assay
Reaction mixtures (2.6 µM nsP2-Trx, 30 µM FRET substreat, 1x PBS pH 7.4, 5mM DTT) were incubated overnight at room temperature. Cleavage product (10-20µL) were separated by SDS-PAGE in 12-wells using 1x diluted NuPage MES running buffer.
The FRET assay in Figure 4c shows a consistent decrease in slope as inhibitor concentration increases. This decrease in slope corresponds with the reading done with just the substrate alone in figure 4a. In figure 4a, we see that both the 430/470 and 430/530 reading are close in proximity and have about the same slope. That is because the FRET substrate CFP-YFP complex is not being cut and the YFP is able to absorb the emission at 470 from CFP and emit at 530 giving us similar slopes. While in the presence of enzyme like in figure 4b we see the two readings appear to grow apart. This is because in the presence of the enzyme the YFP is unable to absorb CFP emission at 470 ultimately not being able to go through with fluorescence energy transfer because it is not within FRET distance of CFP as a result the 530 reading decreases over time. This is due to the enzyme the FRET substrate CFP-YFP complex is not being cut. Being that YFP emits at the 530 ratio and absorbs at the same wavelength CFP emits it is able to conduct energy transfer as it is within FRET distance.
Purification was done in three processes of column chromatography; affinity, ion and size exclusion. This was to be done so that only the protein of interest was present, ultimately substrate or protease being the only factors within in each FRET assay trial. Both our FRET substrate and nsP2 protease were constructed with histidine-tags which allowed for them to bind to nickel in the affinity column. Due the imidazole of the histidine having an affinity to the metal ion within the column it was able to bind while the addition of buffer with increasing imidazole concentration allowed for unwanted lysates to be washed off until. The highest concentration of imidazole buffer eluted the protein of interest. Ion chromatography was used to separate the sample based on charge while size exclusion purified protein further based on size. Protein gel electrophoresis were used to determine how pure a sample was after refinement. FRET assays were conducted on purified FRET substrate and VEEV nsP2 cysteine protease to determine the catalytic activity of the enzyme as well as if inhibitors worked to inhibit cleavage of the FRET substrate.
The FRET assay in Figure 4c shows a consistent decrease in slope as inhibitor concentration increases. This decrease in slope corresponds with the reading done with just the substrate alone in figure 4a. In figure 4a, we see that both the 430/470 and 430/530 reading are close in proximity and have about the same slope. That is because the FRET substrate CFP-YFP complex is not being cut and the YFP is able to absorb the emission at 470 from CFP and emit at 530 giving us similar slopes. While in the presence of enzyme like in figure 4b we see the two readings appear to grow apart. This is because in the prescence of the enzyme the YFP is unable to absorb CFP emission at 470 ultimately not being able to go through with fluorescence energy transfer because it is not within FRET distance of CFP as a result the 530 reading decreases over time. This is due to the enzyme the FRET substrate CFP-YFP complex is not being cut. Being that YFP emits at the 530 ratio and absorbs at the same wavelength CFP emits it is able to conduct energy transfer as it is within FRET distance.
A total of 9 acrylate and vinyl-sulfone based inhibitors were tested in FRET assays against the nsP2 cysteine protease from Venezuelan equine encephalitis virus. Our finding confirmed that some of our acrylate and vinyl-sulfone based inhibitors did work by way of inhibition if nsP2 cysteine protease. Specifically, inhibitors N-7 (acrylate) and N-15 (vinyl sulfone-based) showed high inhibition. Dose-dependent FRET assays and Gel based assays supported this.
Normally when a cysteine protease such as the one on the C-terminal of nsP2 cleaves a substrate their catalytic mechanism involves first the nucleophilic cysteine thiol within the catalytic dyad. It begins when the thiol of the cysteine is deprotonated within its active site by the imidazole of the histidine residue. Next would be the nucleophilic deprotonated anionic sulfur of the cysteine attacks the peptide carbonyl carbon of the substrate, in this case the cleavage junctions of polyprotein nsP1234. In this step, a fragment of the substrate is released with an amine terminus. We can remember that the first cleavage of nsP2 on the polyprotein is at the P3/4 junction. With the release of the peptide fragment the histidine residue is restored and its imidazole is deprotonated again. During this time a thioester intermediate linking the new carboxy-terminus of the substrate to the cysteine thiol. Subsequently, hydrolysis occurs on the thioester bond creating a carboxylic acid moiety on the remaining substrate fragment and regenerating the free enzyme.
In the presence of a high concentration of our competitive inhibitor it can be concluded that when the enzyme acts on it as if it was the polyprotein nsP1234 the enzyme becomes fixed in a covalent bond. This occurs after the deprotonation of the thiol by the imidazole of the histidine residue. The nucleophilic thiol attacks the carbonyl carbon, C4 of our inhibitors and due to electron resonance within our inhibitor become locked in a covalent bond with it. This catalytically deactivates the enzyme from any further polyprotein processing. Ultimately, keeping it from conduct its role as the facilitator of VEEV viral replication.
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