Phenanthridine derivatives as potential HIV-1 protease inhibitors.

BIOMEDICAL REPORTS 13: 66, 2020 Phenanthridine derivatives as potential HIV‑1 protease inhibitors PAVEL V. ERSHOV1,2, YURI V. МEZENTSEV1, LEONID A. KALUZHSKIY1 and ALEXIS S. IVANOV1 1 Federal State Budgetary Institution, V.N. Orekhovich Research Institute of Biomedical Chemistry; Federal State Budgetary Institution, Centre for Strategic Planning and Management of Biomedical Health Risks of The Federal Medical Biological Agency, Moscow 119121, Russia 2 Received April 13, 2020; Accepted September 14, 2020 DOI: 10.3892/br.2020.1373 Abstract. In the present study, the antiviral activity of phen‑ anthridine derivatives was assessed. In total, the inhibitory effect of eight structurally similar low‑molecular‑weight hydrophobic compounds on HIV‑1 protease (HIVp) was investigated. HIVp is a key enzyme in the HIV‑1 life cycle. Surface plasmon resonance technology was used for affinity assessment of compounds binding with either monomeric or dimeric forms of HIVp. HIVp enzyme inhibition assays with chromogenic substrate VII were also used to determine the IC50 values. The most potent compound was 3,3,9,9‑tetrameth yl‑3,4,9,10‑tetrahydro‑2H,8H‑phenanthridine‑1,7‑dione which binds to monomeric and dimeric forms of HIVp (apparent dissociation constant, 2‑7 µM; IC50, 36 µМ), while possessing the most favorable Absorption, Distribution, Metabolism and Excretion parameters. Molecular docking simulations highlighted certain differences in the binding patterns of the phenanthridine derivatives with HIVp amino acid residues forming the flaps domain, monomer/monomer interfaces and the active site cavity of HIVp. Thus, it was hypothesized that the inhibitory effect of phenanthridine compounds on the enzymatic activity of HIVp may be due to restriction of substrate access to the HIVp active site. Introduction Characterization of novel biological activities of chemical compounds is closely associated with identification of poten‑ tial drug prototypes. Phenanthridine derivatives are a group of low‑molecular‑weight (179‑600 Da) non‑peptide organic compounds, which are being actively studied, primarily due to their ability to bind efficiently with DNA and RNA (intercala‑ tors) (1) and also used as dyes (2). Representative compounds of this group have been shown to exhibit significant inhibitory Correspondence to: Dr Pavel V. Ershov, Federal State Budgetary Institution, V.N. Orekhovich Research Institute of Biomedical Chemistry, 10 Building 8, Pogodinskaya, Moscow 119121, Russia E‑mail: Key words: HIV1 protease, phenanthridine derivatives, surface plasmon resonance, monomer, dimer, enzyme inhibition activity against parasitic Leishmania protists (3), as well as activity against MCF‑7, PC3 and HeLa tumor cell lines (4). Moreover, phenanthridine derivatives bind to specific target proteins both in vitro and in vivo, such as Bcl‑XL (5), topoisomerase I (6) and poly (ADP‑ribose) polymerases (7), causing anti‑tumor and anti‑apoptotic effects. Thus, phenan‑ thridines present a class of compounds with a wide (but not fully studied) spectrum of biological and pharmacological properties. The search for novel antiviral drug prototypes, including compounds against HIV‑1, is one of the top priorities of pharmacological screening programs during the initial stages of drug discovery. In the present study, it was hypothesized that phenanthridine derivatives may exhibit biological activity against HIV‑1, since one of the derivatives, 2,3,6,8‑tetra‑ chlorophenanthridine, has been characterized as a HIV‑1 protease (HIVp) dimerization inhibitor, as shown in our previous studies (8,9). The aim of the present study was to assess the anti‑ viral activity of eight new phenanthridine derivatives with homologous structures to 2,3,6,8‑tetrachlorophenanthridine on HIVp. This viral enzyme is an obligatory dimer serving a key role in the HIV‑1 life cycle, however a high rate of mutation during HIV‑1 replication reduces the efficacy of chemotherapy (10‑12). Bioinformatics (structure‑activity relationship predictions and molecular docking simulations), surface plasmon resonance (SPR) and enzyme inhibition assays were used to assess the activities of the derivatives. At least one of the new phenanthridine derivatives, 3,3,9,9‑tetramethyl‑3,4,9,1 0‑tetrahydro‑2H,8H‑phenanthridine‑1,7‑dione (compound 2a), showed different affinities for the monomeric and dimeric forms of HIVp. Docking estimations with the lowest binding energy corresponded to the positioning of compound 2a in two regions of HIVp, the active site cavities and the flaps domain. Thus, it was suggested that binding of compound 2a restricted substrate access to the active site of HIVp due to the inhibitory effect of this compound. Materials and methods Recombinant proteins and chemical compounds. The purity of the recombinant HIVp (Baсhem Holding AG) was >95% as determined by SDS‑PAGE. A micrOTOF‑Q II (Bruker Corporation) mass‑spectrometer was used as an additional 2 ERSHOV et al: PHENANTHRIDINE DERIVATIVES AS HIV-1 PROTEASE INHIBITORS quality control check of the protein preparation according to standard protocols of protein identification (data not shown) (13,14). Chromogenic substrate VII, acetyl‑pepstatin (a known competitive inhibitor of HIVp) (15) and the ‘inter‑ facial’ hexapeptide Palmitoyl‑Thr‑Val‑Ser‑Tyr‑Glu‑Leu were obtained from Bachem Holding AG. A set of phenan‑ thridine derivatives (Table I) was obtained from Asinex and Sigma‑Aldrich; Merck KGaA. Protease inhibitors and phen‑ anthridine derivatives were dissolved in 100% DMSO. Stock solutions were stored at ‑20˚C. 1‑ethyl‑3‑(3‑dimethylaminopro pyl)‑carbodiimide HCl, N‑hydroxysuccinimide and 1M etha‑ nolamine HCl (pH 8.5) were obtained from GE Healthcare. Maleic acid was obtained from Sigma‑Aldrich; Merck KGaA. Absorption, distribution, metabolism and excretion (ADME) and activity prediction for compounds. For prediction of ADME properties, the online resource SwissADME (swis‑ sadme.ch) (16) was used. Prediction of the activity spectrum for the phenanthridine derivatives against key HIV‑1 enzymes (integrase, protease and reverse transcriptase) was performed using HIVprotI (bioinfo.imtech.res.in/manojk/hivproti/) (17). SPR. Biacore 3000 and 8K biosensors (GE Healthcare) were used for registration of intermolecular interactions. HIVp protein was covalently immobilized on the surface of a standard CM5 sensor chip (GE Healthcare) in sodium maleate buffer (pH 6.0) using a standard amine coupling protocol according to the manufacturer's protocol. The procedures for obtaining monomeric and dimeric forms of HIVp on the optical chip were performed as described previously (8,9). Sodium acetate buffer (0.1 M, pH 5.0) containing 1 mM EDTA and 3% DMSO (v/v) was used as the running buffer. A biosensor channel without immobilized HIVp protein was used as a reference channel for subtraction of non‑specific binding to the chip surface. The biosensor signal was recorded in resonance units (RU); one RU approx (...truncated)