Fragment‐Based Screen against HIV Protease

Abstract

We have employed a fragment‐based screen against wild‐type (NL4‐3) HIV protease (PR) using the Active Sight fragment library and X‐ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co‐crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3–1.3 Å resolution. Fragment binding induces a distinct conformation and specific crystal form of TL‐3 inhibited PR during co‐crystallization. One fragment, 2‐methylcyclohexanol, binds in the ‘exo site’ adjacent to the Gly 16 Gly 17 Gln 18 loop where the amide of Gly 17 is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys 14 and Leu 63 . Another fragment, indole‐6‐carboxylic acid, binds on the ‘outside/top of the flap’ via hydrophobic contacts with Trp 42 , Pro 44 , Met 46 , and Lys 55 , a hydrogen bond with Val 56 , and a salt‐bridge with Arg 57 . 2‐acetyl‐benzothiophene also binds at this site. This study is the first fragment‐based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor‐bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target.

Highly Resistant HIV-1 Proteases and Strategies for Their Inhibition

“…Two alternative binding sites were identified in a fragment based crystallographic screen of HIV protease: the loop formed by Gly16-Gly17-Gln18, and a shallow groove on the outside of the flap formed by Trp42, Pro44, Met46, Lys55, Val56, Arg57 [73]. Remarkably, DRV was observed bound to an almost identical location of the flap in crystal structures of the highly resistant PR20 mutant [45].…”

Advantages of crystallographic fragment screening: Functional and mechanistic insights from a powerful platform for efficient drug discovery

“…Soaking at higher concentrations of DMSO can either cause the crystal lattice to collapse or have a detrimental effect on X-ray diffraction. Incorporating DMSO into the protein crystallization condition can circumvent sensitivity of protein crystals to DMSO (Perryman et al, 2010). The cryoprotective properties of DMSO (Atwell et al, 2010) can be advantageous as it allows for a single solution for fragment soaking and cryoprotection (Bauman et al, 2013 a,b).…”

“…This approach cannot identify weak fragment binders since high concentration of fragments can interfere with crystallization. Perryman et al (2010) used co-crystallization approach to identify fragment binding to HIV-1 protease after soaking individual fragments into two different crystal forms failed. Co-crystallization screening at 10 and 2.5 mM fragment concentrations using a crystallization condition for a P6 1 22 crystal form of HIV-1 protease produced two new crystal forms, resulting in the identification of three fragment hits.…”

Molecular Dynamics Study on Function of 2-ylacetic acid-Benzothiophene in Binding of HIV-1 Protease and Inhibitor

“…PDB 中包含 PR、 3TL(抑制剂)、 2FX、 DMS(2-甲基亚砜)、 BME(β-巯基乙醇)和结晶水, 其中, DMS 和 BME 作为有 机溶剂起溶解和稀释作用 [7] , 因此只提取 PR 和 3TL 作 为体系 1, PR, 3TL 和 2FX 作为体系 2. 对于 PR, 先把 B 链残基 Asp25 单质子化 [17] ,…”