Naphthoxazepine inhibitors of HIV-1 integrase: synthesis and biological evaluation

Two sets of compounds derived from the fusion of a diversely annulated naphthoxazepinedione system with 1,3-thiazole and 1,3-oxazole are described. These compounds are close analogues of previously reported thiazolothiazepine inhibitors of human immunodeficiency virus type 1 integrase (HIV-1 IN). Some of the new derivatives show potency similar to that of the reference compounds, thus gaining further insight into the structure-activity relationship of this class of IN inhibitors.     

Allosteric inhibitor development targeting HIV-1 integrase

HIV-1 integrase (IN) is one of three essential enzymes for viral replication, and is a focus of ardent antiretroviral drug discovery and development efforts. Diligent research has led to the development of the strand-transfer-specific chemical class of IN inhibitors, with two compounds from this group, raltegravir and elvitegravir, advancing the farthest in the US Food and Drug Administration (FDA) approval process for any IN inhibitor discovered thus far. Raltegravir, developed by Merck & Co., has been approved by the FDA for HIV-1 therapy, whereas elvitegravir, developed by Gilead Sciences and Japan Tobacco, has reached phase?III clinical trials. Although this is an undoubted success for the HIV-1 IN drug discovery field, the emergence of HIV-1 IN strand-transfer-specific drug-resistant viral strains upon clinical use of these compounds is expected. Furthermore, the problem of strand-transfer-specific IN drug resistance will be exacerbated by the development of cross-resistant viral strains due to an overlapping binding orientation at the IN active site and an equivalent inhibitory mechanism for the two compounds. This inevitability will result in no available IN-targeted therapeutic options for HIV-1 treatment-experienced patients. The development of allosterically targeted IN inhibitors presents an extremely advantageous approach for the discovery of compounds effective against IN strand-transfer drug-resistant viral strains, and would likely show synergy with all available FDA-approved antiretroviral HIV-1 therapeutics, including the IN strand-transfer-specific compounds. Herein we review the concept of allosteric IN inhibition, and the small molecules that have been investigated to bind non-active-site regions to inhibit IN function.     

Discovery and structure-activity relationship studies of a unique class of HIV-1 integrase inhibitors

HIV-1 integrase (IN) is an essential enzyme for viral replication and a validated target for the development of drugs against AIDS. Currently there are no approved drugs that target IN. However, new IN inhibitors are under clinical investigation. As more IN inhibitors enter human drug trials, there is a growing need for the design of novel lead compounds with diverse structural scaffolds and promising pharmacokinetic properties to counteract the difficulties observed with first-generation IN inhibitors. We have identified a novel class of IN inhibitors through the systematic exploration of structure-activity relationships in a series of linomide analogues. The predicted bound conformation of the most active analogues inside the IN active site also supports the observed structure-activity correlation in this new compound class.     

A novel and versatile method for the synthesis of soluble fullerenated polymers

Summary A new route for the preparation of soluble fullerenated polymer through the reaction of carbanion intermediates of polymers with fullerenes, particularly C₆₀, is demonstrated. Confirmation of the covalent attachment of C₆₀ to the polystyrene backbone is by a variety of techniques such as UV-Vis, FT-IR, TGA, SEM and ¹³C NMR etc. The product, which has a visibly brownish yellow cast when compared with the unreacted polymer, is soluble in some common organic solvents. The thermal stability of pure polystyrene is enhanced by C₆₀-chemical modification, and no bonds within the carbon sphere are broken.     

An amphiphilic conjugate approach toward the design and synthesis of betulinic acid-polyphenol conjugates as inhibitors of the HIV-1 gp41 fusion core formation

Exploration of potent inhibitors of the HIV-1 gp41 fusion core formation is a promising strategy to discover small-molecule HIV-1 entry inhibitors for the treatment of HIV-1 infection. In this paper, a series of novel betulinic acid-polyphenol conjugates was designed, guided by molecular modeling of the binding of betulinic acid (BA) and phenolic galloyl/caffeoyl groups in the groove on the gp41 N-terminal heptad repeat (NHR) trimeric coiled coil. These conjugates were synthesized via conjugation of galloyl and caffeoyl groups with BA at the C-28 position. Their inhibitory activities of HIV gp41 six-helix bundle (6-HB) formation between the NHR peptide N36 and the C-terminal heptad repeat (CHR) peptide C34 were evaluated with size-exclusion HPLC. Conjugates bearing a galloyl group were found to exhibit four to sixfold higher inhibitory activities than that of parent compound BA, suggesting that they may be exploitable as HIV-1 fusion/entry inhibitors targeting gp41. The docking study on BA and its derivatives suggests that hydrophobic and hydrogen-bonding pockets exist in the groove of the gp41 NHR trimeric coiled coil and that a potent inhibitor should have amphiphilic structures to cooperatively interact with both pockets. This possibility was explored by incorporating both lipophilic and hydrophilic groups into the conjugates in a well-defined orientation to bind with both pockets in the gp41 NHR-trimer.     

基于结构的HIV-1整合酶抑制剂的虚拟筛选

目的虚拟筛选基于结构的HIV-1整合酶抑制剂。方法从PDB(Protein Data Bank)下载HIV-1整合酶催化核心结构域与LEDGF/p75整合酶结合结构域(integrase binding domain,IBD)的晶体结构(PDB ID:2B4J),通过AutoDockTools对结构进行处理;从ZINC数据库下载化合物结构,用PyRx处理和转换成pdbqt格式,建立一个处理后的化合物数据库;以HIV整合酶为靶点,通过新的虚拟筛选工具PyRx运行AutoDock Vina,对ZINC数据库的化合物进行虚拟筛选;分析得到的小分子抑制剂与整合酶之间的结合情况,并用PyMol对小分子抑制剂与整合酶的结合模式进行3D建模。结果经3轮筛选,发现5个高活性的HIV-1整合酶抑制剂ZINC9486894、ZINC47636331、ZINC57383520、ZINC68964708、ZINC73549421;5个小分子抑制剂与整合酶之间的结合主要是氢键结合力和疏水相互作用。结论通过PyRx运行AutoDock Vina,从ZINC数据库的化合物中筛选出5个新的HIV-1整合酶抑制剂。     

A versatile synthesis of isomeric hyperbranched polyetherketones

Summary Hyperbranched polyetherketones with opposing fluoro and hydroxy terminal groups have been synthesised from the corresponding AB₂ monomers. The choice of A and B in these monomer units has been shown to be important and affects properties such as molecular weight, solubility, and glass transition temperature.     

Chemical synthesis and expression of the HIV-1 Rev protein

The HIV-1 Rev protein is responsible for shuttling partially spliced and unspliced viral mRNA out of the nucleus. This is a crucial step in the HIV-1 lifecycle, thus making Rev an attractive target for the design of anti-HIV drugs. Despite its importance, there is a lack of structural, biophysical, and quantitative information about Rev. This is mainly because of its tendency to undergo self-assembly and aggregation; this makes it very difficult to express and handle. To address this knowledge gap, we have developed two new highly efficient and reproducible methods to prepare Rev in large quantities for biochemical and structural studies: 1) Chemical synthesis by using native chemical ligation coupled with desulfurization. Notably, we have optimized our synthesis to allow for a one-pot approach for the ligation and desulfurization steps; this reduced the number of purification steps and enabled the obtaining of desired protein in excellent yield. Several challenges emerged during the design of this Rev synthesis, such as racemization, reduced solubility, formylation during thioester synthesis, and the necessity for using orthogonal protection during desulfurization; solutions to these problems were found. 2) A new method for expression and purification by using a vector that contained an HLT tag, followed by purification with a Ni column, a cation exchange column, and gel filtration. Both methods yielded highly pure and folded Rev. The CD spectra of the synthetic and recombinant Rev proteins were identical, and consistent with a predominantly helical structure. These advances should facilitate future studies that aim at a better understanding of the structure and function of the protein.     

Design, synthesis and biological evaluation of novel inhibitors of Trypanosoma brucei pteridine reductase 1

Genetic studies indicate that the enzyme pteridine reductase?1 (PTR1) is essential for the survival of the protozoan parasite Trypanosoma brucei. Herein, we describe the development and optimisation of a novel series of PTR1 inhibitors, based on benzo[d]imidazol-2-amine derivatives. Data are reported on 33 compounds. This series was initially discovered by a virtual screening campaign (J. Med. Chem., 2009, 52, 4454). The inhibitors adopted an alternative binding mode to those of the natural ligands, biopterin and dihydrobiopterin, and classical inhibitors, such as methotrexate. Using both rational medicinal chemistry and structure-based approaches, we were able to derive compounds with potent activity against T.?brucei PTR1 (K(i)(app)=7?nM), which had high selectivity over both human and T.?brucei dihydrofolate reductase. Unfortunately, these compounds displayed weak activity against the parasites. Kinetic studies and analysis indicate that the main reason for the lack of cell potency is due to the compounds having insufficient potency against the enzyme, which can be seen from the low K(m) to K(i) ratio (K(m)=25?nM and K(i)=2.3?nM, respectively).     

合成1-氨基环丁甲酸的新方法

以乌洛托品为催化剂,向1-溴代环丁甲酸的异丙醇溶液中通入氨气,在回流条件下制备出1-氨基环丁甲酸。较原文献所载方法不仅合成路线比较短,而且操作也更容易,具有良好的应用前景。     

Crystal structures of novel allosteric peptide inhibitors of HIV integrase identify new interactions at the LEDGF binding site

An optimised method of solution cyclisation gave us access to a series of peptides including SLKIDNLD (2). We investigated the crystallographic complexes of the HIV integrase (HIV-IN) catalytic core domain with 13 of the peptides and identified multiple interactions at the binding site, including hydrogen bonds with residues Thr125 and Gln95, that have not previously been described as being accessible within the binding site. We show that the peptides inhibit the interaction of lens epithelium-derived growth factor (LEDGF) with HIV-IN in a proximity AlphaScreen assay and in an assay for the LEDGF enhancement of HIV-IN strand transfer. The interactions identified represent a potential framework for the development of new HIV-IN inhibitors.     

Targeting the open-flap conformation of HIV-1 protease with pyrrolidine-based inhibitors

HIV protease is a well-established drug target in antiviral chemotherapy. Immense research efforts have been made to discover effective inhibitors, thus making the enzyme one of the most studied and best characterized proteins. Although the protease exhibits high flexibility, all approved drugs target virtually the same protein conformation. The development of viral cross-resistance demands the generation of inhibitors with novel scaffolds and deviating modes of binding. Herein we report the design and the short, high-yielding stereoselective synthesis of a series of chiral, symmetric pyrrolidine-based inhibitors targeting the open-flap conformation of the protease. The obtained co-crystal structure with one derivative provides a valuable starting point for further inhibitor design.     

阻垢剂性能评定方法及新设备开发

对污垢的准确监测是促进阻垢剂产业发展的关键技术.可用于评价阻垢剂性能的方法很多,介绍了有关研究者对传统方法进行的改进,以及提出的一些新方法、新技术.合理选择评定方法,对于了解不同阻垢剂的性能与作用机理、开发更加有效的阻垢剂是非常重要的.文章对现有阻垢剂性能评定方法进行了简要综述,着重介绍了所开发的新装置及应用实例.     

Design, synthesis and analysis of novel bicyclic and bifunctional protease inhibitors

Two novel synthetic inhibitors were designed to combine the advantageous properties of Bowman Birk inhibitor (BBI) and sunflower trypsin inhibitor-1 (SFTI-1). As is the case for BBI, the novel inhibitors have two active sites that give dual independent protease inhibition. However, they also possess a small bicyclic structure, reminiscent of the single-site SFTI-1. It is found that the synthetic inhibitors retain the potent inhibitory properties of the parent structures; they are also found to be relatively resistant to proteolysis. Their inhibition properties and a comparison of their stability to proteolysis relative to SFTI-1 are described. It is found that the new inhibitors do indeed allow bifunctional inhibition, although, unlike BBI, the small size of the inhibitor prevents the simultaneous inhibition of two proteases at the same time.     

Heterogeneity in recombinant HIV-1 integrase corrected by site-directed mutagenesis: the identification and elimination of a protease cleavage site

Purified recombinant human immunodeficiency virus type 1 (HIV-1) integrase and certain deletion mutants exhibit heterogeneity consistent with proteolysis at a site close to the C-terminus. Electrospray ionization mass spectrometric analysis indicated that proteolytic cleavage generated a protein missing five residues from the C-terminus. PCR mutagenesis of amino acids on either side of the cleavage site identified two changes which were subsequently shown to prevent clipping when proteins were expressed and purified from Escherichia coli: the substitution of Arg284, the residue on the C-terminal side of the cleavage site, by either glycine or lysine. The introduction of either of these mutations into full-length integrase did not affect in vitro 3' processing or strand transfer activities. Thus, the incorporation of either of these mutations is likely to be beneficial when homogeneity of HIV-1 integrase is a concern, as in crystallographic or nuclear magnetic resonance spectroscopic experiments.      

A beta-1,4-galactosyltransferase from Helicobacter pylori is an efficient and versatile biocatalyst displaying a novel activity for thioglycoside synthesis

Helicobacter pylori is a highly persistent and common pathogen in humans. It is the causative agent of chronic gastritis and its further stages. HP0826 is the beta-1,4-galactosyltransferase involved in the biosynthesis of the LPS O-chain backbone of H. pylori. Though it was first cloned nearly a decade ago, there are surprisingly limited data about the characteristics of HP0826, especially given its prominent role in H. pylori pathogenicity. We here demonstrate that HP0826 is a highly efficient and promiscuous biocatalyst. We have exploited two novel enzymatic activities for the quantitative synthesis of the thiodisaccharide Gal-beta-S-1,4-GlcNAc-pNP as well as Gal-beta-1,4-Man-pNP. We further show that Neisseria meningitidis beta-1,4-galactosyltransferases LgtB can be used as an equally efficient catalyst in the latter reaction. Thiodisaccharides have been extensively used in structural biology but can also have therapeutic uses. The Gal-beta-1,4-Man linkage is found in the Leishmania species LPG backbone disaccharide repeats and cap, which have been associated with vector binding in Leishmaniasis.