Biomedical subjects

Usman Bacha

Publications and source records attributed to Usman Bacha.

3 recordsLinked to original sources

PubMed2006

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.

Allosteric Site↗

PubMed2004

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.

Amino Acid Sequence↗

PubMedDate not supplied

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.

Drug Resistance, Viral↗