Despite a high current standard of care in antiretroviral therapy for HIV, multidrug-resistant strains continue to emerge, underscoring the need for additional novel mechanism inhibitors that will offer expanded therapeutic options in the clinic. We report a new class of small molecule antiretroviral compounds that directly target HIV-1 capsid (CA) via a novel mechanism of action. The compounds exhibit potent antiviral activity against HIV-1 laboratory strains, clinical isolates, and HIV-2, and inhibit both early and late events in the viral replication cycle. We present mechanistic studies indicating that these early and late activities result from the compound affecting viral uncoating and assembly, respectively. We show that amino acid substitutions in the N-terminal domain of HIV-1 CA are sufficient to confer resistance to this class of compounds, identifying CA as the target in infected cells. A high-resolution co-crystal structure of the compound bound to HIV-1 CA reveals a novel binding pocket in the N-terminal domain of the protein. Our data demonstrate that broad-spectrum antiviral activity can be achieved by targeting this new binding site and reveal HIV CA as a tractable drug target for HIV therapy.

A method for estimating structural properties of random media is described. The size, number density, and scattering strength of particles are estimated from an analysis of the radio frequency (rf) echo signal power spectrum. Simple correlation functions and the accurate scattering theory of Faran [J.J. Faran, J. Acoust. Soc. Am. 23, 405-418 (1951)], which includes the effects of shear waves, were used separately to model backscatter from spherical particles and thereby describe the structures of the medium. These methods were tested using both glass sphere-in-agar and polystyrene sphere-in-agar scattering media. With the appropriate correlation function, it was possible to measure glass sphere diameters with an accuracy of 20%. It was not possible to accurately estimate the size of polystyrene spheres with the simple spherical and Gaussian correlation models examined because of a significant shear wave contribution. Using the Faran scattering theory for spheres, however, the accuracy for estimating diameters was improved to 10% for both glass and polystyrene scattering media. It was possible to estimate the product of the average scattering particle number density and the average scattering strength per particle, but with lower accuracy than the size estimates. The dependence of the measurement accuracy on the inclusion of shear waves, the wavelength of sound, and medium attenuation are considered, and the implications for describing the structure of biological soft tissues are discussed.

Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large in the order of ϳ1.4 MDa. We now demonstrate that dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M r values of ϳ1.4 MDa and ϳ700 kDa. Kinetic analysis demonstrates that in vitro the ϳ700-kDa complex is produced more rapidly than the ϳ1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide or N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the ϳ700-kDa Apaf-1 containing apoptosome complex was predominately formed. This complex processed effector caspases. Thus, the ϳ700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis.

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