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 regula on, 60 and confer decreased suscep bility to SM111 SM111 Recent advances in HIV-1 an retroviral therapy (ART) have substan ally 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 selec on 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 an viral drugs. HIV-1 accessory proteins, including Vpu, enhance viral replica on NL43 (No drug) 40 2 2 20 102 10 10 and in vivo pathogenesis and thus may be a rac ve targets for new classes of an viral drugs. 30 NL43 (100uM SM111) 40 100 CD4 1 1 101 10 10 CD317 (Tetherin) 20 50 0 0 0 0
% Infected cells - Day 6 - cells Day % Infected 10 10 10 20 0 1 2 3 4 0 1 2 3 4 Vpu promotes virion release by downregula ng the host restric on 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 cells - Day 6 - cells Day % Infected 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 ac vity in HIV-1 replica on remains unproven. A puta ve inhibitor of Vpu viroporin ac vity 0 0
1 2 3 4 5 6 6 - cells Day % Infected 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 Relative expression to GFP only SM111 concentration 100uM 1 1 10 10 101 WT-Vpu We inves gated the an -HIV-1 ac vity of a novel acylguanidine compound, SM111, which has Drug concentration Relative expression to GFP only 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 Hepa s 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) 20 based small molecule pa ent-derived subtype B Pol sequences were used to cells % Infected 20 20 20 SM111A % Infected cells % Infected ii) BIT225 is an acylguanidine-based compound under assess an viral ac vity of each drug. Details of 4 strains SM111C + 100uM SM111 6 Day - cells Infected % 0 inves ga on 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 muta ons 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 muta ons on Tetherin (CD317)/CD4 downregula on ac vity, viral spread and HIV resistant strain Drug resistance mutations doses) was added at 24hrs. Nega ve (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-infec on. B, % NRTI-RS (AZT) D67N,T69N,K70R,T215Y days 2-6 by flow cytometry. B, HIV-1 replica on 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 replica on is not affected by SM113, an vi) Raltegravir (RAL) is an integrase inhibitor (INI) PI-RS (IDV) M46L,I54V,I84V,L90M impaired down regula on ac vity by all SM111-selected mutants. C, Vpu mutant viruses displayed INI-RS (RAL) N155H acylguanidine analog that has no an -viroporin ac vity. 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 suscep bility to Cells Inhibi on of HIV replica on in SupT1 cells (cell line lacks BST2/CD317-Tetherin) in a mul cycle 8 day assay. SM111, with the 5AA dele on (SM111A) remaining most sensi ve, trunca on (SM111C) moderately A GFP reporter CD4+ T cell line (CEM-GXR) and primary PBMCs were used in mul -cycle replica on assays to E, Inhibi on of HIV replica on in human primary PBMCs, by 50uM SM111 in a mul cycle 9 day assay. 60 sensi ve, and I17R subs tu on (SM111H) least sensi ve to the drugs an viral ac vity. 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 quan fied 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 quan fy viral growth in PBMCs. CEM-GXR cells were also transfected 20
with WT Vpu or Vpu encoding SM111-selected muta ons, and downregula on 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 cells - Day 6 - cells Day % Infected 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 replica on 30 NRTI-RS 40 WT (NL43) Δ DelVpuNL43 PI-RS SM111 (100uM) 40 No drug 50 ! ! INI-RS ! ! Drug (SM111) p24 (pg/ml) NL43 20
1. CEM cells Day$2$ cells Infected % 0.29$%$ 20 20 $ 20 ! cells % Infected ! !! Day$3$ 0.45$%$
% Infected cells % Infected 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 6 - cells Day % Infected 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, Replica on SM111 inhibits HIV-1 viral egress p24 by Elisa kine cs of recombinant NL43 strains encoding resistance muta ons 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 respec ve 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) titer Viral 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 % BIT225 100uM 15 Strain I cells harvested at 48hrs post-transfec on with pNL4-3 displayed an SM111 dose-dependent decrease. Viral 60 20 No Drug Strain J Strain K ter was measured using CEM-GXR cells. B, Supernatant p24 concentra ons of WT and mutant HIV-1 50 0 Strain L Strain C 0 20 40 60 80 100 0 2 4 6 10 Strain M
% Infected cells par cles released from HEK293 cells 48hrs post transfec on under different concentra ons of SM111. Days post infection Strain N (truncation) Drug concentration (uM) Cell count relative to No drug (%) C D 5 Conclusions
100 100 0 0 5 10 v SM111 is a novel acylguanidine-based small molecule with broad an viral ac vity Days post infection 50 50 v SM111 displays an -HIV-1 ac vity with minimal cytotoxicity in T cell lines and PBMCs B HIV-1 Vpu: v 0 SM111 inhibits the replica on of HIV-1 strains resistant to NRTI, NNRTI, PI and INI drugs, 0 Relative viability to 0.1% DMSO NL43 MQPIIVAIVA LVVAIIIAIV VWSIVIIEYR ..! SM111A ------XXXXX------..! Relative viability to 0.1% DMSO 0.1% to viability Relative 10uM 25uM 50uM indica ng a dis nct mechanism of ac on 100uM 10uM 25uM 50uM 100uM SM111 concentration SM111C ------* ! Greene et al, An viral Research 2008 SM111 concentration SM111H ------R------.. ! v SM111 selects for muta ons located in the HIV-1 Vpu TM domain that impair Tetherin Figure 4. Muta ons in the Vpu transmembrane (TM) domain are selected in the presence of SM111. and CD4 down regula on ac vity Figure 1. Effects of SM111 on cell viability and prolifera on. A, CEM-GXR cells were incubated with various A, SM111 resistant HIV-1 variants were generated by passaging NL43 in the presence of 100uM drug. B concentra ons of SM111. Cell counts and viability were determined using ViaCount (Millipore). No toxicity v Mutant HIV-1 strains and strains lacking Vpu remain at least par ally sensi ve to SM111, and C, Unique Vpu muta ons were observed in three outgrowth strains, resul ng in either a 5 AA was observed with up to 100uM SM111 or SM113 (an SM111 variant with no viroporin ac vity), while indica ng that addi onal host and/or viral target(s) may be required for drug ac vity dele on (strain A), a trunca on (strain C) or a subs tu on I17R (strain H) of the TM sequence. BIT225 was cytotoxic at >20uM. B, SM111 had minimal effects on cell prolifera on in a 6 day assay. C, SM111 is non-toxic in HEK-293 cells up to 100uM. D, SM111 was non toxic in PBMCs up to 50uM; some v SM111 represents a promising lead molecule for future ART studies toxicity was observed at 100uM.