Binding assays

MicroBioSpin 6 columns were used to measure the binding of [3H]BMS-626529 to gp120. Binding solutions (30 μl) containing 25 mM Tris-HCl (pH 7.5), 125 mM NaCl, 50 nM gp120JRFL, and serial dilutions of [3H]BMS-626529 were allowed to equilibrate and then adsorbed to a MicroBioSpin 6 column. The column was centrifuged (14,000 rpm) for 5 min, the eluent was collected, and radioactivity was determined with a scintillation counter. To measure dissociative kinetics, 150 nM [3H]BMS-626529 was incubated with 60 nM gp120 at ambient temperature for 1 h to achieve equilibrium binding, and then a large molar excess (14-fold) of soluble CD4 protein was added to drive dissociation. Aliquots were taken at the indicated time intervals, adsorbed to a spin column, and centrifuged, and the radioactivity in the eluent was quantitated. Comparison of the tritium signal from parallel samples with and without the soluble CD4 challenge allowed for the determination of the percent compound bound.

Cell lines

PBMCs infected with HIV-1 clinical isolates; MT-2 and PM1 cells

Preparation method

Soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

6 days; dissolved in 100% dimethyl sulfoxide (DMSO) and serially diluted to the desired concentration such that the final DMSO concentration in cell culture assays was 1%.

Applications

BMS-626529 exhibits low cytotoxicity in several cell types from different human tissues such as MT-2 (T lymphocytes), HEK293 (kidney), PM1 and PBMCs. BMS-626529 exhibits EC50 value against the CXCR4-tropic LAI virus of 0.7 nM and also exhibits broad spectrum of activity.

Patients

Adults (aged ≥18 years) infected with subtype B HIV-1.

Dosage form

600 mg or 1200mg with or without 100 mg ritonavir; 8 days; orally administrated.

Applications

BMS-663068, the prodrug of BMS-626529, reduces plasma HIV-1 RNA levels and increases median absolute CD4+ T-cell counts. Also, BMS-663068 is well tolerated. BMS-626529 has favorable pharmacokinetics following administration of the prodrug BMS-663068.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

EC50<10 nM against the vast majority of viral isolates

HIV-1 attachment inhibitors represent a new class of entry inhibitors that prevent the initial interaction between virus and host cell by binding to the viral envelope protein gp120 and blocking attachment of the virus to the CD4 receptor on CD4+ T-cells. BMS-626529 is a novel small-molecule attachment inhibitor that targets HIV-1 gp120 and prevents its binding to CD4+ T-cells.

In vitro: The activity of BMS-626529 is virus dependent, due to heterogeneity within gp120. BMS-626529 had half-maximal effective concentration values of<10 nm against the vast majority of viral isolates; however, susceptibility varied by>6 log10, with half-maximal effective concentration values in the low pM range against the most susceptible viruses. Measurement of the binding affinity of BMS-626529 for purified gp120 suggests that a contributory factor to its inhibitory potency may be a relatively long dissociative half-life [1].

In vivo: No animal in-vivo data available currently

Clinical trial: BMS-663068 is a prodrug of the small-molecule inhibitor BMS-626529. The maximum median decreased in plasma HIV-1 RNA load from baseline ranged from 1.21 to 1.73 log10 copies/mL. Plasma concentrations of BMS-626529 were not associated with an antiviral response, while low baseline inhibitory concentrations and the minimum and average steady-state BMS-626529 plasma concentrations, when adjusted by the baseline protein binding–adjusted 90% inhibitory concentration, were linked with antiviral response. BMS-663068 was generally well tolerated [2].

References:
[1] Nowicka-Sans B, Gong YF, McAuliffe B, Dicker I, Ho HT, Zhou N, Eggers B, Lin PF, Ray N, Wind-Rotolo M, Zhu L, Majumdar A, Stock D, Lataillade M, Hanna GJ, Matiskella JD, Ueda Y, Wang T, Kadow JF, Meanwell NA, Krystal M.  In vitro antiviral characteristics of HIV-1 attachment inhibitor BMS-626529, the active component of the prodrug BMS-663068. Antimicrob Agents Chemother. 2012;56(7):3498-507.
[2] Nettles RE, Schürmann D, Zhu L, Stonier M, Huang SP, Chang I, Chien C, Krystal M, Wind-Rotolo M, Ray N, Hanna GJ, Bertz R, Grasela D.  Pharmacodynamics, safety, and pharmacokinetics of BMS-663068, an oral HIV-1 attachment inhibitor in HIV-1-infected subjects. J Infect Dis. 2012;206(7):1002-11.