Novel furocoumarins as potential HIV-1 integrase inhibitors

A series of seven novel, rationally designed N-substituted 3-{3,5-dimethylfuro[3,2-g]coumarin-6-yl}propanamides have been prepared as potential HIV-1 integrase (IN) inhibitors via a five-step pathway commencing with resorcinol and diethyl 2-acetylglutarate, and the HIV-1 IN inhibition potential of these compounds has been examined relative to raltegravir, a known HIV-1 IN inhibitor.     

4-Hydroxy-5-pyrrolinone-3-carboxamide HIV-1 integrase inhibitors

The viral enzyme integrase is essential for the replication of HIV-1 and, after the discovery of Isentress™, represents a validated target for anti-retroviral therapy. Incorporation of the dihydroxycarbonyl pharmacophore into a pyrrolinone scaffold led to the discovery of 5-pyrrolinone-3-carboxamides as a structurally diverse class of HIV-1 integrase inhibitors.     

Design of inhibitors of the HIV-1 integrase core domain using virtual screening

Acquired immunodeficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiencyvirus (HIV). The integrase (IN) enzyme of HIV interacts with several cellular and viral proteins during the integration process.Thus, it represents an appropriate target for antiretroviral drugs (ARVs). We performed virtual screening of database compoundsand designed analogues using Elvitegravir (EVG) as a standard compound. The 378 screened compounds were retrieved fromZINC, ChemSpider, PubChem, and ChemBank Chemical Databases based on chemical similarity and literature searches related tothe structure of EVG. The Physiochemical properties, Bioactivity, Toxicity and Absorption, Distribution, Metabolism and Excretionof Molecules (ADME) of these compounds were predicted and docking Experiments were conducted using Molegro VirtualDocker software. The docking and ADME suggested very significant results in regard to EVG. The MolDock and Rerank scoreswere used to analyze the results. The compounds ZINC26507991 (-84.22), Analogue 9 (-68.49), ZINC20731658 (-66.79),ZINC00210363 (-43.44) showed better binding orientation with IN receptor model with respect to EVG (182.52). The ZINC26507991has showed significant ADME result.     

Development of a high-throughput assay for the HIV-1 integrase disintegration reaction

Both HIV-1 integrase (IN) and the central catalytic domain of IN (IN-CCD) catalyze the disintegration reaction in vitro. In this study, IN and IN-CCD proteins were expressed and purified, and a high-throughput format enzyme-linked immunosorbent assay (ELISA) was developed for the disintegration reaction. IN exhibited a marked preference for Mn²⁺ over Mg²⁺ as the divalent cation cofactor in disintegration. Baicalein, a known IN inhibitor, was found to be an IN-CCD inhibitor. The assay is sensitive and specific for the study of disintegration reaction as well as for the in vitro identification of antiviral drugs targeting IN, especially targeting IN-CCD.     

Enzymatic capability of HIS-tagged HIV-1 integrase using oligonucleotide disintegration substrates

Disintegration, wherein a half-site integration substrate is resolved into separate viral and host DNA components via DNA strand transfer, is one of three well-established in vitro activities of HIV-1 integrase. The role of disintegration in the HIV-1 replicative cycle, however, remains a mystery. In this report, we describe the expression inEscherichia coli and purification of HIV-1 integrase as a fusion protein containing a 6×His tag at its amino terminus. Integrase resolved dumbbell and Y-substrates optimally at pH 6.8–7.2 in the presence of 2 mM MnCl₂. Substrate requirements for intramolecular disintegration included a 10 base pair viral U5 LTR arm and a CA dinucleotide located at the 3 end of the LTR. Disintegration was not sensitive to changes in the host DNA portion of the substrate. A dumbbell substrate with a 5 oligo-dA tail also underwent disintegration. The released LTR arm with an oligo-dA tail was utilized as a template primer by several DNA polymerases indicating that disintegration occurred via nucleophilic attack on the phosphodiester bond located immediately adjacent to the CA dinucleotide at the 3 end of the LTR. Coupled disintegration-DNA polymerase reactions provided a highly efficient and sensitive means of detecting disintegration activity. Integrase also catalyzed an apparently concerted disintegration-5-end joining reaction in which an LTR arm was transferred from one dumbbell substrate molecule to another.     

Synthesis and evaluation of substituted 4-arylimino-3-hydroxybutanoic acids as potential HIV-1 integrase inhibitors

A series of readily accessible 4-arylimino-3-hydroxybutanoic acids have been prepared and evaluated as potential HIV-1 Integrase inhibitors. None of the ligands exhibited significant toxicity against human embryonic kidney (HEK 293) cells, while five of them showed activity against HIV-1 integrase – the most active (6c) with an IC₅₀ value of 3.5?μM. In silico docking studies indicate the capacity of ligand 6c to interact with several amino acid residues and the two Mg²⁺ cations in the HIV-1 integrase receptor cavity.     

Nitrogen-containing polyhydroxylated aromatics as HIV-1 integrase inhibitors: synthesis,structure-activity relationship analysis,and biological activity

Four series of forty-five nitrogen-containing polyhydroxylated aromatics based on caffeic acid phenethyl ester were designed and synthesized as HIV-1 integrase (IN) inhibitors. Most of these compounds inhibited IN catalytic activities in low micromolar range. Among these new analogues, compounds 9e and 9f were the most potent IN inhibitors with IC₅₀ value of 0.7 μM against strand transfer reaction. Their key structure-activity relationships were also discussed.     

Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: part II. integrase inhibition

We report molecular modeling and functional confirmation of Ole and HT binding to HIV-1 integrase. Docking simulations identified two binding regions for Ole within the integrase active site. Region I encompasses the conserved D64-D116-E152 motif, while region II involves the flexible loop region formed by amino acid residues 140-149. HT, on the other hand, binds to region II. Both Ole and HT exhibit favorable interactions with important amino acid residues through strong H-bonding and van der Waals contacts, predicting integrase inhibition. To test and confirm modeling predictions, we examined the effect of Ole and HT on HIV-1 integrase activities including 3'-processing, strand transfer, and disintegration. Ole and HT exhibit dose-dependent inhibition on all three activities, with EC(50)s in the nanomolar range. These studies demonstrate that molecular modeling of target-ligand interaction coupled with structural-activity analysis should facilitate the design and identification of innovative integrase inhibitors and other therapeutics.     

Active site binding modes of the beta-diketoacids: a multi-active site approach in HIV-1 integrase inhibitor design

Predicting a bioactive conformation of a ligand is of paramount importance in rational drug design. The task becomes very difficult when the receptor site possesses a region with unusual conformational flexibility. Significant conformational differences are present in the active site regions in the available crystal structures of the core domains of HIV-1 integrase (IN). Among all reported IN inhibitors, the β-diketoacid class of compounds has proved to be of most promise and indeed S-1360 was the first IN inhibitor to enter clinical studies. With an aim to predict the bioactive (active site bound) conformation of S-1360, we performed extensive docking studies using three different reported crystal structures where the active site or partial active site region was resolved. For comparison we extended our studies to include 5CITEP (the first compound cocrystallized with IN core domain) and a bis-diketoacid (BDKA). We found that the conformation of S-1360 when bound in one of the active sites matches that of the experimentally observed results of IN escape mutants resistant to S-1360. Therefore, we propose that this active site conformation is the biologically relevant conformation and can be used for the future structure-based drug design studies selectively targeting IN.     

Cellular protein TTRAP interacts with HIV-1 integrase to facilitate viral integration

TTRAP is a PML-NB protein that is involved in the NF-κB signaling pathway. TTRAP was recently identified by yeast two-hybrid analysis as a HIV-1 integrase (HIV-1 IN) interacting protein. This interaction was verified by co-immunoprecipitation, GST pull-down, and intracellular imaging, and deletion assays suggested that the N-terminal 180 residues of TTRAP are responsible for the interaction. In stable TTRAP knock-down cell lines, the integration of viral vectors decreased significantly compared with non-silenced cell lines. Conversely, overexpression of TTRAP by transient transfection increased the percentage of integration events. This is the first time that TTRAP has been shown to interact with HIV-1 IN and facilitate lentiviral vector integration. These findings reveal a new function of TTRAP and expand our understanding of the cellular response to HIV infection. The interaction between TTRAP and HIV-1 IN may be useful in designing new anti-viral strategies as well as for improving the efficiency of lentiviral-vector-mediated gene delivery.     

Design and discovery of 5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide inhibitors of HIV-1 integrase

Raltegravir (RAL) is a first clinically approved integrase (IN) inhibitor for the treatment of HIV but rapid mutation of the virus has led to chemo-resistant strains. Therefore, there is a medical need to develop new IN inhibitors to overcome drug resistance. At present, several IN inhibitors are in different phases of clinical trials and few have been discontinued due to toxicity and lack of efficacy. The development of potent second-generation IN inhibitors with improved safety profiles is key for selecting new clinical candidates. In this article, we report the design and synthesis of potent 5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide analogues as second-generation IN inhibitors. These compounds satisfy two structural requirements known for potent inhibition of HIV-1 IN catalysis: a metal chelating moiety and a hydrophobic functionality necessary for selectivity against the strand transfer reaction. Most of the new compounds described herein are potent and selective for the strand transfer reaction and show antiviral activity in cell-based assays. Furthermore, this class of compounds are drug-like and suitable for further optimization and preclinical studies.     

Constructing HIV-1 integrase tetramer and exploring influences of metal ions on forming integrase-DNA complex

HIV-1 integrase (IN) is essential for the replication of HIV-1 in human cells. At present, the complete structure of complex IN-DNA has not been resolved. In this paper, a HIV-1 IN tetramer model was built with homology modeling and molecular dynamics simulation approach, in which two Mg2+ ions were reasonably located in each catalytic core domain. Moreover, it was found that the AB and CD chains of HIV-1 IN tetramer were different in the structures and metal ions of HIV-1 IN tetramer might have great influences on DNA locating on IN. These findings may provide a more complete structural basis for guiding drug discovery and revealing integration mechanism.     

Structure-based amelioration of PXR transactivation in a novel series of macrocyclic allosteric inhibitors of HIV-1 integrase

Previous studies have identified a series of imidazo[1,2-a]pyridine (IZP) derivatives as potent allosteric inhibitors of HIV-1 integrase (ALLINIs) and virus infection in cell culture. However, IZPs were also found to be relatively potent activators of the pregnane-X receptor (PXR), raising the specter of induction of CYP-mediated drug disposition pathways. In an attempt to modify PXR activity without affecting anti-HIV-1 activity, rational structure-based design and modeling approaches were used. An X-ray cocrystal structure of (S,S)-1 in the PXR ligand binding domain (LBD) allowed an examination of the potential of rational structural modifications designed to abrogate PXR. The introduction of bulky basic amines at the C-8 position provided macrocyclic IZP derivatives that displayed potent HIV-1 inhibitory activity in cell culture with no detectable PXR transactivation at the highest concentration tested.     

HIV-1整合酶及其抑制剂的研究进展

HIV-1整合酶是由HIV病毒pol基因编码的分子量为32KD的蛋白质,是HIV病毒复制的必需酶之一,它催化病毒DNA整合入宿主染色体DNA。人类细胞中没有HIV 整合酶的类似物[1],理论上抑制整合酶对人体副作用很小。因此HIV-1整合酶成为继HIV-1蛋白酶,逆转录酶后治疗艾滋病的富有吸引力和合理的靶标。本文综述了HIV整合酶结构,抑制剂的研究以及以HIV-1 整和酶为靶点治疗AIDS方法的最新研究进展。     

Anti-HIV-1 integrase diterpenoids from Dichrocephala benthamii

Five new clerodane diterpenoid dichrocephnoids A–E (1,2, 6–8) together with three known analogues were isolated from the whole herb of Dichrocephala benthamii C.B. Clarke. Their structures were elucidated on the basis of spectroscopic analyses (UV, IR, MS, 1D and 2D NMR). The relative configurations of the new compounds were established by NOESY correlations, and the absolute configuration of 1 was determined on the basis of CD methods. Dichrocephnoid A (1) possesses a very rare pentacyclic ring system with highly oxygenated functionalities. All of the isolated compounds exhibited inhibitory activity against HIV-1 integrase, however, dichrocephnoid C (6) showed the most promising inhibition of the enzyme with IC₅₀ value of 12.35 ± 1.27 μM.     

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.     

Synthesis and biological evaluation of 5-fluoroquinolone-3-carboxylic acids as potential HIV-1 integrase inhibitors

A series of new quinolone-3-carboxylic acids as HIV-1 integrase inhibitors featuring a fluorine atom at C-5 position were synthesized and evaluated for their antiviral activity in C8166 cell culture. These newly synthesized compounds showed anti-HIV activity against wild-type virus with an EC₅₀ value ranging from 29.85 to 0.032 μΜ. The most active compound 4e exhibited activity against wild-type virus and the mutant virus A17 with an EC₅₀ value of 0.032 and 0.082 μΜ, respectively. Preliminary structure–activity relationship of these 5-fluoroquinolone-3-carboxylic acids was also investigated.     

Design and synthesis of novel pyrimidone analogues as HIV-1 integrase inhibitors

A series of novel pyrimidone analogues have been designed and synthesized as HIV-1 integrase (IN) inhibitors. This study demonstrated that introducing a substituent in the N1-position of the pyrimidone scaffold does not significantly influence IN inhibitory activity. Molecular docking studies showed these compounds could occupy the IN active site and form pi–pi interactions with viral DNA nucleotides DC16 and DA17 to displace reactive viral DNA 3′OH and block intasome activity.