Mutation of Asn28 disrupts the dimerization and enzymatic activity of SARS 3CL(pro) .

Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL(pro), is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL(pro) and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 A from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CL(pro). Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K(d). The crystallographic structure of the N28A mutant determined at 2.35 A resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL(pro).

New developments for the design, synthesis and biological evaluation of potent SARS-CoV 3CL(pro) inhibitors.

A series of trifluoromethyl, benzothiazolyl or thiazolyl ketone-containing peptidic compounds as SARS-CoV 3CL protease inhibitors were developed and their potency was evaluated by in vitro protease inhibitory assays. Three candidates had encouraging results for the development of new anti-SARS compounds.

Development of broad-spectrum halomethyl ketone inhibitors against coronavirus main protease 3CL(pro).

Coronaviruses comprise a large group of RNA viruses with diverse host specificity. The emergence of highly pathogenic strains like the SARS coronavirus (SARS-CoV), and the discovery of two new coronaviruses, NL-63 and HKU1, corroborates the high rate of mutation and recombination that have enabled them to cross species barriers and infect novel hosts. For that reason, the development of broad-spectrum antivirals that are effective against several members of this family is highly desirable. This goal can be accomplished by designing inhibitors against a target, such as the main protease 3CL(pro) (M(pro)), which is highly conserved among all coronaviruses. Here 3CL(pro) derived from the SARS-CoV was used as the primary target to identify a new class of inhibitors containing a halomethyl ketone warhead. The compounds are highly potent against SARS 3CL(pro) with K(i)'s as low as 300 nM. The crystal structure of the complex of one of the compounds with 3CL(pro) indicates that this inhibitor forms a thioether linkage between the halomethyl carbon of the warhead and the catalytic Cys 145. Furthermore, Structure Activity Relationship (SAR) studies of these compounds have led to the identification of a pharmacophore that accurately defines the essential molecular features required for the high affinity.

Inhibition of HIV-2 protease by HIV-1 protease inhibitors in clinical use.

Over the past 10 years, protease inhibitors have been a key component in antiretroviral therapies for HIV/AIDS. While the vast majority of HIV/AIDS cases in the world are due to HIV-1, HIV-2 infection must also be addressed. HIV-2 is endemic to Western Africa, and has also appeared in European countries such as Portugal, Spain, and Estonia. Current protease inhibitors have not been optimized for treatment of HIV-2 infection; therefore, it is important to assess the effectiveness of currently FDA-approved protease inhibitors against the HIV-2 protease, which shares only 50% sequence identity with the HIV-1 protease. Kinetic inhibition assays were performed to measure the inhibition constants (K(i)) of the HIV-1 protease inhibitors indinavir, nelfinavir, saquinavir, ritonavir, amprenavir, lopinavir, atazanavir, tipranavir, and darunavir against the HIV-2 protease. Lopinavir, saquinavir, tipranavir, and darunavir exhibit the highest potency with K(i) values of 0.7, 0.6, 0.45, and 0.17 nm, respectively. These K(i) values are 84, 2, 24, and 17 times weaker than the corresponding values against the HIV-1 protease. In general, inhibitors show K(i) ratios ranging between 2 and 80 for the HIV-2 and HIV-1 proteases. The relative drop in potency is proportional to the affinity of the inhibitor against the HIV-1 protease and is related to specific structural characteristics of the inhibitors. In particular, the potency drop is high when the maximum cap size of the inhibitors consists of very few atoms. Caps are groups located at the periphery of the molecule that are added to core structures to increase the specificity of the inhibitor to its target. The caps positioned on the HIV-1 protease inhibitors affect selectivity through interactions with distinct regions of the binding pocket. The flexibility and adaptability imparted by the higher number of rotatable bonds in large caps enables an inhibitor to accommodate changes in binding pocket geometry between HIV-1 and HIV-2 protease.

Long-range cooperative interactions modulate dimerization in SARS 3CLpro.

Severe acute respiratory syndrome (SARS) is an infectious disease caused by the human coronavirus, SARS-CoV. The main viral protease, SARS 3CLpro, is a validated target for the development of antiviral therapies. Since the enzyme is a homodimer and the individual monomers are inactive, two approaches are being used to develop inhibitors: enzyme activity inhibitors that target the active site and dimerization inhibitors. Dimerization inhibitors are usually targeted to the dimerization interface and need to compete with the attractive forces between subunits to be effective. In this paper, we show that the dimerization of SARS 3CLpro is also under allosteric control and that additional and energetically more favorable target sites away from the dimerization interface may also lead to subunit dissociation. We previously identified a cluster of conserved serine residues (Ser139, Ser144, and Ser147) located adjacent to the active site of 3CLpro that could effectively be targeted to inactivate the protease [Bacha, U et al. (2004) Biochemistry 43, 4906-4912]. Mutation of any of these serine residues to alanine had a debilitating effect on the catalytic activity of 3CLpro. In particular, the mutation of Ser147, which does not make any contact with the opposing subunit and is located approximately 9 A away from the dimer interface, totally inhibited dimerization and resulted in a complete loss of enzymatic activity. The finding that residues away from the dimer interface are able to control dimerization defines alternative targets for the design of dimerization inhibitors.

Synthesis of glutamic acid and glutamine peptides possessing a trifluoromethyl ketone group as SARS-CoV 3CL protease inhibitors.

Trifluoromethyl-ß-amino alcohol 11 [(4S)-tert-butyl 4-amino-6,6,6-trifluoro-5-hydroxyhexanoate] was synthesized in five steps starting from Cbz-l-Glu-OH 5 where the key step involved the introduction of the trifluoromethyl (CF3) group to oxazolidinone 7, resulting in the formation of silyl ether 8 [(4S,5S)-benzyl 4-(2-(tert-butoxycarbonyl)ethyl)-5-(trifluoromethyl)-5-(trimethylsilyloxy)oxazolidine-3-carboxylate]. Compound 11 was then converted into four tri- and tetra-glutamic acid and glutamine peptides (1-4) possessing a CF3-ketone group that exhibited inhibitory activity against severe acute respiratory syndrome coronavirus protease (SARS-CoV 3CLpro).

Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro.

SARS (severe acute respiratory syndrome) is caused by a newly discovered coronavirus. A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CL(pro) (also termed SARS-CoV 3CL(pro)). We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CL(pro) as well as a truncated form containing only the catalytic domains. The recombinant proteins have been characterized enzymatically using a fluorescently labeled substrate; their structural stability in solution has been determined by differential scanning calorimetry, and novel inhibitors have been discovered. Expression of the catalytic region alone yields a protein with a reduced catalytic efficiency consistent with the proposed regulatory role of the alpha-helical domain. Differential scanning calorimetry indicates that the alpha-helical domain does not contribute to the structural stability of the catalytic domains. Analysis of the active site cavity reveals the presence of subsites that can be targeted with specific chemical functionalities. In particular, a cluster of serine residues (Ser139, Ser144, and Ser147) was identified near the active site cavity and was susceptible to being targeted by compounds containing boronic acid. This cluster is highly conserved in similar proteases from other coronaviruses, defining an attractive target for drug development. It was found that bifunctional aryl boronic acid compounds were particularly effective at inhibiting the protease, with inhibition constants as strong as 40 nM. Isothermal titration microcalorimetric experiments indicate that these inhibitors bind reversibly to 3CL(pro) in an enthalpically favorable fashion, implying that they establish strong interactions with the protease molecule, thus defining attractive molecular scaffolds for further optimization.

Protease inhibition in African subtypes of HIV-1.

Of the 42 million people infected with HIV-1 worldwide, 30 million are in Africa. However, the HIV-1 subtypes prevalent in Africa are not the same that are prevalent in North America and Western Europe. In these developed regions, subtype B is responsible for the vast majority of HIV infections, whereas in sub-Saharan Africa subtypes A and C, and to a lesser extent subtype G, account for most of the infections. These subtypes exhibit genomic differences as large as 30% with respect to subtype B. These differences involve current drug targets, including the HIV-1 protease. Since protease inhibitors have been developed and tested against the HIV-1 B subtype, and proteases from other subtypes carry up to ten amino acid polymorphisms, it is important to assess the influence of these naturally occurring polymorphisms on the potency of existing inhibitors, as well as their synergistic interactions with mutations known to cause drug resistance. This review will examine the effects of naturally occurring polymorphisms on the efficacy of current protease inhibitors and the effects of well characterized drug-resistant mutations within the framework of non-B subtypes. At the biochemical level, non-B-subtype polymorphisms lower the binding affinities of existing clinical inhibitors, but not to the point of causing drug resistance. However, these polymorphisms amplify the effects of mutations causing drug resistance and may play a role in the long-term viability of these inhibitors.