Synthesis And Screening Of HIV Entry Inhibitors Target On Gp120

Background and ObjectionAn estimated34million people are infected worldwide with human immuno-deficiency virus (HIV), the etiologic cause of acquired immunodeficiency syndrome (AIDS). The AIDS epidemic becomes more and more serious in the world. The successful development of a safe and effective HIV vaccine is still in the future. Therefore, research continues to focus on disease treatment by chemical anti-HIV agents.Until now,24chemical entities and9compositions were approved in clinical. They consist of highly active antiretroviral therapy (HAART), protease inhibtor (PR), integrase inhibitor and virus entry inhibitor. On HAART and PR regimens, multiple drug therapies can lead to increased adverse effects and toxicities due to long-term use and drug-drug interactions. Only3chemical entities of last two classes were approved.Among these targets, viral entry is one of the most promising for HIV drug development. HIV entry inhibitor, although only2chemical entities were approved, was found effective for the virus resistant to HARRT and PR therapy. Enfuvirtide (T-20), which is a HIV fusion inhibitor, was the first drug with a target other than RT and PR to be approved by the US FDA. The success of enfuvirtide and maraviroc validates the clinical application of viral entry inhibitors as a new class of antiretroviral drugs. Continued effort in discovering new HIV entry inhibitors, especially potent, orally bioavailable small molecules, is still needed.The progress that HIV entry into host cells involves at least three steps:(1) first, an attachment step, this step requires CD4receptor binding,(2) second, co-receptor binding,(3) third, a fusion process, HIV fuse with target cellular membrane. The envelope glycoprotein (Env) on the surface of virus mediates the entry progress. Virus can attach to target cells, followe by specific binding of HIV gp120to the CD4receptor on the cellular membrane. This binding induces a conformational change in gp120that opens up a high-affinity binding site located within the third variable loop (V3) and surrounding surfaces for the chemokine co-receptors (primarily CCR5and CXCR4). Co-receptor binding results in further conformational rearrangements of gp120that expose the fusion-peptide domain of gp41. The heptad repeat (HR) regions, HR1and HR2of the three subunits of gp41, fold and pack into a six-helix bundle, which brings the viral and cell membranes into juxtaposition and creates pores in the target cell membrane, enabling the release of viral capsid into the cytoplasm.From the virus fuse target cells progress, different entry inhibitors can be divided into3classes:(1) first, gp120-CD4binding inhibitors,(2) second, co-receptor binding inhibitors, and (3) fusion inhibitors, especially inhibit gp41core structure formation. Each step of this sequential entry process has been suggested as a potential target for developing anti-HIV-1drugs. Among HIV entry into host cells progress, an attachment step that gp120binds to CD4was the first step. HIV entry inhibitors can target this first step can theoretically reduce the harmful to human.The binding of HIV-1gp120to the cellular receptor CD4is critical for HIV-1 entry into cells and has also been suggested as a potential target for developing anti-HIV-1therapy. HIV entry inhibitors that block gp120-CD4interactions mainly consist of two types of compounds, large molecular compounds and small molecular compounds. Large molecular inhibitors were sCD4and neutralization antibodies and small molecular inhibitors consist of BMS-378806and NBD-556analogues. The Phe43cavity has been suggested as the putative binding site of BMS-378806and NBD-556, potent entry inhibitors. This discovery is fundamentally important and raised hopes that small molecule inhibitors are capable of disrupting protein-protein interactions when targeted to the so-called "hot spots" such as the cavity.Zhou et al isolated two antibodies, which have broad neutralization of diverse viral strains from immunoglobulin genes from individual cells. They were the mostly broad neutralization antibodies which can neutralize over90%of circulating HIV-1isolates. The crystal structure of VRC01in complex with a human immunodeficiency virus HIV-1gp120core showed that VRC01partially mimics CD4interaction with gp120. A shift from the CD4-defined orientation, however, focuses VRC01onto the vulnerable site of initial CD4attachment, allowing it to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. V5loop is wedged in the gap formed by the heavy and light chains of VRC01. Arg61in the CDR H2penetrates into the cavity formed by gp120V5and β24, locking V5into a less flexible conformation. In contrast, CD4only interacts with the "front side" of V5. Heavy chain Arg61goes behind the V5and provides5hydrogen bonds to the cavity formed by gp120V5and β24. These results provide a foundation for rational vaccine design that is based not only on the particular mode of antibody-antigen interaction but also on defined relationships between genomic antibody precursors, somatic hypermutation, and interface-recognition element. It also built a new target for designing anti HIV gp120 compounds. Until now, there is no report that small molecular compounds bind to this hot-spot.BMS-378806is the most potent small molecule inhibitor reported so far that blocks the interaction between gp120and CD4with IC505nmol/L. BMS-378806enter clinical I research and the further clinical investigation was suspended because of its narrow antiviral range. BMS-488043enters clinical II, and it was also suspended because of large dose. Until now, there are no BMS-378806analogues to be approved.We screened a drug-like small molecule chemical library consisting of approximately33,000compounds using a syncytium formation assay and identified two N-phenyl-N-piperidin-4-yl-oxalamide analogs which significantly inhibited syncytium formation. Both NBD-556and NBD-557showed potent inhibitory activity against gp120-CD4interaction. These two compounds interfere with the gpl20-CD4interaction by binding to unliganded gp120core, resulting in inhibiting HIV-1particles from attaching to the cellular receptor, the first step of HIV entry into target cells. NBD-556and NBD-557did not inhibit gp41six-helix bundle formation, nor interact with co-receptors. Development of a new HIV-1entry inhibitor is importantBased on the above reported in the literature, we begin our research from the following two parts:(1) Virtual screening method was used for screening the compound library based on Arg61. Then, we calculate the difference of the difference binding free energy of gp120with higher score compounds. The compounds with higher difference of binding free energy were purchased and their anti HIV activities were also examined. The lead compound NC-2was obtained, and its mechanism of action was also examined. The structure of active compounds were also been modified in order to get more active compounds. (2) Synthesis and activity of compounds which block the gp120-CD4interaction using NBD-556as lead compound were studied.Methods and materialsThe crystal structure of VRC01in complex with a human immunodeficiency virus HIV-1gp120core showed that its broad neutralization had a relationship with its complex structure, especially with the hydrogen bonds that Arg61penetrating into the V5and β24cavity. We use MM/PBSA method to calculate the difference binding free energy of gp120and Arg61or58-61amino acid residues. Virtual screening method was used for screening the compound library based on binding site1and2. Then, we calculate the difference of the difference binding free energy of gp120and higher score compounds. The compounds with higher difference of binding free energy were purchased and their anti HIV activities were also examined. The lead compound NC-2was obtained, and its mechanism of action was also examined.(1) Ligand database, ibs2009oct_ncReceptor,3NGB-ABinding site, site1, Arg61, site258to61amino acid residues(2) Generate protomol files with surflex-dock. Virtual screen the ibs2009oct_nc compound library.(3) Calculate the difference of free binding energy of high scored compounds and binding site using MM/PBSA method.(4) Purchase the compounds of larger difference of free binding energy for activity test.(5) Lead compound NC-2was obtained. It has good inhibitory acitivities on the infection of pseudotyped HIV-1JRFL and HIV-1mB virus. The mechanism of action was also studied.(6) Analogues based on NC-2were synthesized, and the activities were examined.The activities of reported NBD-556analogues did not significantly improve compare to NBD-556. We designed and synthesized NBD-556analogues and examined their inhibition activities againt HIV infection. The residues of2,2,6,6-tetramethyl-piperidine and oxalamine were remained. The substituent was introduced on R position; it maybe substituted aromatic group or aromatic heterocyclic compound. The synthetic route is the addition of substituted aniline to ethyl chlorooxoacetate in the presence of base or amidation reaction using aniline and diethyloxalate. Analogues based on NBD-556were synthesized, and the activities were examined.ResultsVirtual screening the ibs2009oct_nc natural compound library with Arg61and58to61amino acid residues as two binding sites was proceeded. Firstly, generate protomol files with surflex-dock using12CPU calculate, other parameters were default values.113compounds with docking scores greater than8.0which bind site1were obtained.180compounds with docking scores greater than8.0which bind site2were obtained. Among them,30compounds can bind both site1and site2. We choose these30compounds and calculate the difference of free binding energy.We calculated the difference of free binding energy of high scored compounds and gp120with MM/PBSA method. Compare them with ΔGgP120-VRC01and ΔGgp120-CD4. ΔGgp120-Arg61, PBTOT=-10.67, GBTOT=-13.89.ΔGgp120-(58-61), PBTOT=-18.37, GBTOT=-16.27。ΔGgp120-(58-61) smaller than ΔGgp120-Arg61showed that the affinity of gp120bind to58-61amino acid residues is stronger than that of gp120with Arg61. ΔGgp120-VRC01, PBTOT=-113.62, GBTOT=-78.88. ΔGgp120-CD4, PBTOT=-57.95, GBTOT=-23.77. The results of ΔGgp120-VRC01smaller than ΔGgp120-CD4also showed that the affinity of gp120bind to VRC01is stronger than that of gp120bind to CD4. So VRC01has good neutralization ability.Purchase the compounds with higher difference of binding free energy and examine their anti HIV activities. A Total of19molecules that showed higher difference of binding free energy were screened from40000molecules. Among them, NC-2showed anti-HIV-1activities against the infection of HIV-1pseudotyped virus and laboratory-adapted HIV-1. The IC50values of NC-2for inhibiting infection against HIV-1ⅢB and pseudotyped HIV-1JRFL were1.95±0.44μM and10.58±0.13μM, respectively. The CC50values of NC-2for inhibit U87.CD4.CCR5cell and MT-2cell were121.25μM and17.23μM, respectively. NC-12has a similar IC50and CC50values, so it inhibiting ability may be caused by its cytotoxicity.NC-2could block HIV-1envelope mediated cell-cell fusion. The results of ELISA suggested that NC-2could inhibit the binding of HIV-1gp120to CD4but it could not block the formation of gp41six-helix bundle in vitro.We designed and synthesized two types of analogues based on NC-2.9analogues were obtained. Synthesis of another series of compounds is ongoing.The synthetic route is the addition of substituted aniline to ethyl chlorooxoacetate in the presence of base or amidation reaction using aniline and diethyloxalate.8analogues were obtained, and the inhibition activities against HIV infection showed that the activities of compounds did not greatly improve after structural modification.ConclusionThis computer virtual screening with virus inhibition activity testing method was used for screening small molecular against the infection of HIV. A small molecular NC-2was obtained. The results of mechanism of action showed that NC-2was a HIV entry inhibitor and its binding target was HIV Env. NC-2can block the binding of HIV-1gp120and CD4, but it could not block the formation of gp41six-helix bundle in vitro. The Arg61binding site may be an important target of small molecular compounds against HIV infection. This method may be used for large scale screening HIV-1entry inhibitors targeting gp120.The researches on analogues synthesized base on NC-2are ongoing.9of analogues were obtained. Another series of analogues also had breakthrough progress.The structure of NBD-556analogues was optimized.8analogues were synthesized. The inhibition activities against HIV infection showed that the activities of compounds did not greatly improve after structural modification.