BMS-707035

• 产品号: S1366
• CAS号: 729607-74-3
• 分子式: C17H19FN4O5S
• 分子量: 410.4199632
• 保 存: -20°C

产品别名

• 标题: BMS-707035
• IUPAC标准名: 2-(1,1-dioxo-1$l^{6},2-thiazinan-2-yl)-N-[(4-fluorophenyl)methyl]-5-hydroxy-1-methyl-6-oxo-1,6-dihydropyrimidine-4-carboxamide
• IUPAC传统名: 2-(1,1-dioxo-1$l^{6},2-thiazinan-2-yl)-N-[(4-fluorophenyl)methyl]-5-hydroxy-1-methyl-6-oxopyrimidine-4-carboxamide

产品登记号

• CAS号: 729607-74-3

产品性质

• 作用靶点: Integrase
• 成盐信息: Free Base
• 溶解度: DMSO
• 保存条件: -20°C

产品详细信息

详细说明 (English)

Research Area

• Description: Cancer

Biological Activity

• Description: BMS-707035 is a specific HIV-I integrase (IN) inhibitor with IC50 of 15 nM.
• Targets: HIV-I integrase (IN)
• IC50: 15 nM ^[1]
• In Vitro: BMS-707035 is a pyrimidine carboxamide similar to Raltegravir, the first integrase inhibitor licensed for clinical use. BMS-707035 is a potent, specific, and reversible HIV-I integrase (IN) inhibitor that blocks HIV IN strand transfer activity with IC50 of 15 nM. ^[1]However, several IN mutations, including V75I, Q148R, V151I, and G163R are found to confer resistance to HIV IN inhibitors. The binding of BMS-707035 and target DNA to IN are mutually exclusive events, as revealed by the fact that the inhibition of strand transfer catalysis by BMS-707035 is overcome by increasing amount of target DNA. The binding affinity of BMS-707035 to IN is also affected by the four terminal bases at the 5’ end of the pre-processed U5 long terminal repeat (LTR). Gln^148 of IN is crucial for the binding of BMS-707035 to IN. ^[1]The 3’ terminus of the viral LTR, on the other hand, retards the rate of BMS-707035 association with IN, by regulating the kinetics of binding and dissociation. ^[2]
• Clinical Trials: A Phase II clinical trial of BMS-707035 for HIV-1 has been withdrawn.

Protocol

• Protocol: Kinase Assay ^[1]
• Determination of Strand Transfer Activity and Inhibition: The in vitro activities of purified INs in combination with the various duplex LTRs are measured through a scintillation proximity assay (SPA). In a first step, the viral LTR duplexes are prepared by annealing individual oligonucleotides. The viral (donor) LTR DNA is then attached, via a 5’-biotin linker on the plus strand, to streptavidin-coated SPA PVT beads as follows. SPA PVT beads (10 mg) are suspended in 0.2 mL of PBS. The suspension is then centrifuged at <5000 ×="" g="" for="" 15="" min.="" the="" supernatant="" is="" removed,="" and="" the="" pellet="" is="" resuspended="" with="" 0.2="" ml="" of="" pbs,="" 0.85="" m="" nacl,="" and="" 21="" μl="" of="" 12="" μm="" duplex="" hiv="" ltr="" dna.="" the="" sequences="" of="" the="" duplexes="" are="" as="" follows,="" except="" for="" the="" variations="" in="" the="" underlined="" bases:="" plus="" strand,="" 5’-biotin-acccttttagtcagtgtggaaaatctctagca;="" minus="" strand,="" 5’-actgctagagattttccacactgactaaaag.="" the="" ltr="" dna="" is="" allowed="" to="" bind="" for="" 60="" min="" at="" room="" temperature="" with="" gentle="" rocking,="" after="" which="" time="" 0.8="" ml="" of="" te="" is="" added.="" the="" mixture="" is="" then="" centrifuged="" at=""><5000 ×="" g="" and="" resuspended="" in="" 0.8="" ml="" of="" te,="" 50="" mm="" nacl.="" the="" beads="" are="" washed="" 4="" additional="" times="" with="" te,="" 50="" mm="" nacl,="" each="" time="" centrifuging="" to="" remove="" unbound="" viral="" ltr="" dna.="" the="" final="" pellet="" is="" resuspended="" in="" 0.2="" ml="" of="" pbs="" and="" stored="" at="" 4="" °c="" before="" use.="" enzyme="" complexes="" for="" 80="" strand="" transfer="" reactions="" are="" prepared="" as="" follows:="" 0.15="" ml="" of="" bead-dna="" complexes,="" 2.25="" ml="" of="" spa="" buffer="" (13.3="" mm="" dithiothreitol,="" 32="" mm="" mops,="" ph="" 7.0,="" 0.067%="" np-40,="" 6.4%="" polyethylene="" glycol,="" 25.6="" mm="">₂, 12.8% (v/v) Me₂SO, and 100 mM NaCl), and IN (37 μg of WT, 88 μg of N155H, and 36 μg of Q148R) are incubated at 37 °C. After 1.5 hours, complexes are pelleted and resuspended with 2.4 mL of SPA buffer. The proper amount of each IN is determined through titration experiments and represented the minimal amount of enzyme required to produce the maximal amount of strand transfer products. The target DNA is prepared (5’-[^33P]TGACCAAGGGCTAATTCACT-3’ annealed to 5’-[^33P]AGTGAATTAGCCCTTGGTCA-3’) in a separate step by individually 5’ end labeling each of the oligonucleotides with [γ-^33P]ATP. The ^33P-labeled oligonucleotides are then annealed to form the target duplex DNA. Strand transfer assays consists of combining 30 μL of IN complexes with 10 μL of 25% Me₂SO/H₂O) (v/v) in white microtiter plates. After 10 min of incubation at 37 °C, 10 μL of ^33P-labeled target DNA (1 × 10^6 cpm) is added (final concentration of target is 0.92 nM). Plates are returned to 37 °C for 2 hours, after which time the strand transfer reactions are stopped by the addition of 200 μL of PBS, 50 mM EDTA. Plates are allowed to stand overnight before reading on a Topcount scintillation counter. HIV-1 IN complexes are evaluated for inhibitor sensitivity using the SPA assay, except that 10 μL of a 5-fold serial dilution of BMS-707035 in 25% (v/v) Me₂SO/H₂O is added in the place of 10 μL of 25% (v/v) Me₂SO/H₂O. Data are analyzed by fitting to a sigmoidal dose-response curve.

References

• References: [1] Dicker IB, et al. J Biol Chem, 2007, 282(43), 31186-31196.
• References: [2] Langley DR, et al. Biochemistry, 2008, 47(51), 13481-13488.

参考文献

• Dicker IB, et al. J Biol Chem, 2007, 282(43), 31186-31196.
• Langley DR, et al. Biochemistry, 2008, 47(51), 13481-13488.