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Inhibition of HIV-1 Replication by a Novel Acylguanidine-Based Molecule

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Inhibition of HIV-1 Replication by a Novel Acylguanidine-Based Molecule Inhibition of HIV-1 Replication by a Novel Acylguanidine-based Molecule Philip Mwimanzi1, Ian Tietjen2, Aniqa Shahid1, Scott C. Miller2, David Fedida2, Zabrina Brumme1,3, Mark A Brockman1,3,4 1Faculty of Health Sciences Simon Fraser Univ; 2Dept of Anesthesiology, Pharmacology and Therapeutics, Univ of British Columbia; 3BC Centre for Excellence in HIV/AIDS; 4Dept of Molecular Biology and Biochemistry, Simon Fraser Univ Background and Objec;ve SM111 inhibits in vitro HIV-1 replica;on SM111-selected muta;ons impair Vpu-mediated Tetherin and CD4 down regulaon, 60 and confer decreased suscep;bility to SM111 SM111 Recent advances in HIV-1 an1retroviral therapy (ART) have substan1ally reduced morbidity and B 60 A C SM113 Uninfected WT-NL43 ΔVpu-NL43 4 4 50 A 104 10 10 B 60 mfi:400 mortality, but the selec1on and transmission of drug-resistant strains necessitates ongoing 40 SM111 mfi:149 mfi:622 100 CD4 3 3 40 SM113 103 10 10 CD317 (Tetherin) discovery of new an1viral drugs. HIV-1 accessory proteins, including Vpu, enhance viral replicaon NL43 (No drug) 40 2 2 20 102 10 10 and in vivo pathogenesis and thus may be arac1ve targets for new classes of an1viral drugs. 30 NL43 (100uM SM111) 40 100 CD4 1 1 101 10 10 CD317 (Tetherin) 20 50 0 0 0 0 % Infected% cellsDay - 6 10 10 10 20 0 1 2 3 4 0 1 2 3 4 Vpu promotes virion release by downregulang the host restric1on factor BST-2/Tetherin. Vpu is 20 100 101 102 103 104 10 10 10 10 10 10 10 10 10 10 % Infected cells 10 1uM % Infected% cellsDay - 6 10uM 50 also reported to be a viroporin (i.e. forming channels in cell membranes), but the role of this 100uM SM111A-NL43 SM111C-NL43 SM111H-NL43 4 4 10 10 104 0 Drug concentration (CD317) Tetherin mfi:524 mfi:724 mfi:702 ac1vity in HIV-1 replicaon remains unproven. A putave inhibitor of Vpu viroporin ac1vity 0 0 1 2 3 4 5 6 Infected% cellsDay - 6 3 0 3 10 10 103 1uM Days post infection 10uM (BIT225) has been described; however, it displays high toxicity in T cell cultures. 100uM 2 2 2 3.16uM 17.8uM31.6uM56.2uM 10 10 0 No drug 1uM 10 10uM only GFP to expression Relative SM111 concentration 100uM 1 1 10 10 101 WT-Vpu We inves1gated the an1-HIV-1 ac1vity of a novel acylguanidine compound, SM111, which has Drug concentration only GFP to expression Relative SM111_ASM111_CSM111_HGFP only 0 0 10 10 100 0 1 2 3 4 0 1 2 3 4 WT-Vpu D 100 E 10 10 10 10 10 10 10 10 10 10 100 101 102 103 104 SM111_ASM111_CSM111_HGFP only been shown to inhibit viroporin proteins encoded by Influenza, Dengue, and Hepa1s C viruses. NL43 (No drug) 80 NL43 (No drug) NL43 (100uM SM111) NL43 (50uM SM111) GFP 80 Materials and Methods 60 60 D No drug 60 C SM111A 60 SM111C + No drug 100uM SM111 Drugs Viruses SM111H 40 40 40 i) SM111 (lead compound) is a novel acylguanidine- Wild type NL43 and recombinant NL43 strains encoding 40 p24 (pg/ml) p24 20 based small molecule paent-derived subtype B Pol sequences were used to Infected% cells 20 20 20 SM111A % Infected% cells ii) BIT225 is an acylguanidine-based compound under assess an1viral ac1vity of each drug. Details of 4 strains SM111C + 100uM SM111 % Infected cells - Day 6 0 inves1gaon by Biotron, Ltd (Australia) harbouring major NRTI, NNRTI, PI and INI resistance 0 0 SM111H 2 4 6 8 2 4 6 8 10 0 iii) Zidovudine (AZT) is a nucleoside reverse mutaons are shown in Table 1. Days post infection Days post infection 1 2 3 4 5 6 SM111A SM111C SM111H WT (NL43) transcriptase inhibitor (NRTI) Days post infection Table 1. Patient-derived pol sequences Figure 2. An;-HIV ac;vity of SM111. A, CEM-GXR cells were infected at MOI of 0.3% and SM111 (various iv) Efavirenz (EFV) is a non-nucleoside reverse Figure 5. Effects of Vpu mutaons on Tetherin (CD317)/CD4 downregulaon acvity, viral spread and HIV resistant strain Drug resistance mutations doses) was added at 24hrs. Negave (no drug) wells were used as controls. Viral growth was monitored at transcriptase inhibitor (NNRTI) SM111 resistance. A, Dot plots showing Tetherin cell surface expression at day 6 post-infec1on. B, % NRTI-RS (AZT) D67N,T69N,K70R,T215Y days 2-6 by flow cytometry. B, HIV-1 replicaon was inhibited by SM111 in a dose-dependent manner. v) Indinavir (IDV) is a protease inhibitor (PI) NNRTI-RS (EFV) K103N, V108I,M230L Tetherin and CD4 expression on CEM T cells transfected with wild-type Vpu or Vpu mutants demonstrates Results are displayed as % infected cells at day 6. C, HIV-1 replicaon is not affected by SM113, an vi) Raltegravir (RAL) is an integrase inhibitor (INI) PI-RS (IDV) M46L,I54V,I84V,L90M impaired down regulaon ac1vity by all SM111-selected mutants. C, Vpu mutant viruses displayed INI-RS (RAL) N155H acylguanidine analog that has no an1-viroporin ac1vity. Results are displayed as % infected cells at day 6. D, enhanced viral spread in the presence of SM111. D, Mutant strains , showed a range of suscep1bility to Cells Inhibi1on of HIV replicaon in SupT1 cells (cell line lacks BST2/CD317-Tetherin) in a mul1 cycle 8 day assay. SM111, with the 5AA dele1on (SM111A) remaining most sensi1ve, truncaon (SM111C) moderately A GFP reporter CD4+ T cell line (CEM-GXR) and primary PBMCs were used in mul1-cycle replicaon assays to E, Inhibi1on of HIV replicaon in human primary PBMCs, by 50uM SM111 in a mul1 cycle 9 day assay. 60 sensi1ve, and I17R subs1tu1on (SM111H) least sensi1ve to the drugs an1viral ac1vity. propagate viruses in the presence or absence of SM111 (0-100μM) or other drugs (AZT, EFV, IDV, or RAL; 0-100nM). Infected CEM-GXR cells were quan1fied by flow cytometry as the % GFP+ events among viable cells SM111 inhibits drug resistant HIV-1 strains 40 HIV-1Δvpu displays par;al resistance to SM111 that differs by cell type in culture, while p24 Elisa was used to quan1fy viral growth in PBMCs. CEM-GXR cells were also transfected 20 with WT Vpu or Vpu encoding SM111-selected mutaons, and downregulaon of CD4 or Tetherin (CD317) NL43 No drug A B C WT No Drug A B WT No drug 80 40 NNRTI-RS-EFV 60 0 60 100 WT-50uM-SM111 % Infected% cellsDay - 6 WT No drug NRTI-RS-AZT No drug ΔVpu No drug) monitored by flow cytomery. 100nM Δ Vpu No drug PI-RS-IDV ΔVpu No drug) WT + 100uM SM111 SM111 (100uM) ΔVpu+ 100uM SM111 Δ Vpu-50uM-SM111 INI-RS-RAL WT+ 100uM SM111 60 NNRTI-RS SM111_ASM111_CSM111_H 40 Vpu+ 100uM SM111 Assessment of viral replicaon 30 NRTI-RS 40 WT (NL43) Δ DelVpuNL43 PI-RS SM111 (100uM) 40 No drug 50 ! ! INI-RS ! ! Drug (SM111) (pg/ml) p24 NL43 20 1. CEM cells Day$2$ % Infected cells 0.29$%$ 20 20 $ 20 ! Infected% cells ! !! Day$3$ 0.45$%$ % Infected% cells 0 Infect GFP-reporter T-cell line with HIV; $ 0 0 Day$4$ 2 4 6 2 4 6 8 2 4 6 8 10 0.84$%$ 0 monitor viral spread by flow cytometry 10 Infected% cellsDay - 6 Days post infection Flowcytometry $ Days post infection Days post infection Culture in the Day$5$ Viral infection 3.12$%$ (0.003 MOI) presence or absence ! PI-RS NL43 Figure 6. NL4-3 Δvpu exhibits suscep;bility to SM111 that is dependent in part on cell type. A, NL4-3 Day$6$ INI-RS of drugs 0 $ 10.6$%$ Δvpu displayed minimal resistance to SM111 in CEM-GXR cells but was more resistant to drug in SupT1 cells !! !! 2 4 6 NNRTI-RSNRTI-RS GFP+$ 2. Human PBMCs !! !! Days post infection Viral isolates as shown in (B). C, NL4-3 Δvpu replicated poorly in PBMC and displayed minimal resistance to SM111. Collect supernatant every 3 days; Measure Figure 3: SM111 is inhibits replica;on of NRTI, NNRTI, PI and INI resistant HIV-1 strains. A, Replicaon SM111 inhibits HIV-1 viral egress p24 by Elisa kine1cs of recombinant NL43 strains encoding resistance mutaons to current ARVs in the presence of the PHA activation Viral infection Culture in the 07 2500 for 72hr (0.003 MOI) presence or absence respec1ve ARV or SM111. B, Spread of drug resistant HIV-1 strains and wild type NL43 control in the A 1.0×10 B WT-NL43 of drugs Delta Vpu absence or presence of SM111. Results are displayed as % infected cells at day 6. 8.0×1006 2000 SM111A SM111C 06 SM111H 6.0×10 1500 SM111 exhibited limited toxicity in cell lines and PBMCs 4.0×1006 1000 p24ng/ml SM111 selects muta;ons in the transmembrane domain of Vpu 06 Viral titer (vp/ml) 2.0×10 500 NL43 Viability day 2 50 0 A Strain A C 0 A B Strain B 110 25uM 50uM 25 Strain C 100uM 12.5uM 10uM 25uM 50uM SM111 No Drug 100uM 100 100 1uM Strain D Strain H (I17R) No drug SM113 SM111 concentration 90 10uM Strain E SM111 concentration 80 17.8uM 20 Strain F 80 60 31.6uM Strain G Strain A (deletion) 56.2uM Strain H Figure 7. Inhibi;on of HIV-1 release by SM111. A, Titers of NL4-3 virions from the supernatants of HEK293 70 40 Viability % Viability BIT225 100uM 15 Strain I cells harvested at 48hrs post-transfec1on with pNL4-3 displayed an SM111 dose-dependent decrease.
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