Reagents and viruses
HEK293T (HEK, human embryonic kidney cells) were purchased from ATCC (Manassas, VA). The following reagents were obtained from the NIH AIDS Reference and Reagent Program, Division of AIDS, NIAID: HOS CD4+ CXCR4+ Cells from Dr. Nathaniel Landau; pDOLHIVenv from Dr. Eric O. Freed and Dr. Rex Risser; TZM-bl cells from Dr. John C. Kappes, Dr. Xiayun Wu and Tranzyme Inc.; CEM-GFP cells from Dr. Jacques Corbeil; pNL4-3 from Dr. Malcom Martin; and monoclonal antibody 16H3 (ARP-12559) from Duke Human Vaccine Institute. The pNL4-3 Luc AM pseudotyping backbone plasmid was a generous gift from Dr. John Moore. Dr. Joseph Sodroski donated the expression plasmid for HIV-1 HxBc2 strain Envelope. Escherichia coli strains XL-10 gold (Agilent) and Stbl2 cells (Invitrogen) were used for propagating DNA. Thermostable DNA polymerase (PfuUltra™) was obtained from Stratagene Inc. (La Jolla, CA). Phusion® High-Fidelity Polymerase was purchased from New England Biolabs (Ipswich, MA). Integrated DNA Technologies supplied Custom-oligonucleotide primers. Polyethylenimine (PEI) 25 kDa linear polymer, was obtained from Polysciences, Inc. (Warrington, PA). All other reagents were purchased from Sigma-Aldrich unless otherwise specified.
Peptide synthesis
A CEM microwave synthesizer (Liberty Blue) was used for solid phase peptide synthesis of PTs, as previously described [14, 21, 22, 39]. Fmoc-4-azido-Proline used in the synthesis was prepared as previously reported [21]. All other Fmoc-, Boc- protected amino acids, N,Nʹ-diisopropylcarbodiimide, ethyl (hydroxyimino) cyanoacetate (OxymaPure) and rink amide resin (100–200 mesh size, 0.53 meq/g substitution) were purchased from Chem-Impex International, INC. Ethynylferrocene and CuI catalyst for the click reaction, and hydrazine, were purchased from Sigma Aldrich. HPLC purifications were performed using a Waters® HPLC system with reverse phase (RP) C18 prep columns. Purity verification of cPTs was carried out by analytical C18 RP-HPLC column, using a BeckmanCoulter® HPLC system. HPLC grade acetonitrile (ACN), Millipore-MilliQ water and 0.1% TFA were used as solvents for the HPLC purification. Mass was confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Passaging of fully-infectious virus with inhibitor dose escalation
Fully-infectious NL4-3 virus was used for virus passaging studies. Passaging in the presence of inhibitors was performed using a previously published protocol [45] and started at the determined IC50 value of the inhibitor. Culture supernatant was collected and lysed with Triton X-100, either weekly or biweekly, to determine the amount of p24 proteins present in the supernatant. The level of p24, determined by p24 ELISA [16, 23, 38] was dependent on the amount of virus able to be produced in the culture. When the level of p24 in peptide-treated cultures was within threefold comparable to the amount in untreated control cultures, the concentration of inhibitor in the culture was escalated two-fold. Three independent cultures for each treatment were carried out in 48-well plates using CEM-GFP cells [46] as the target cells for virus infection. Each plate only contained one treatment to prevent virus contamination between sample groups. Phosphate buffered saline (PBS) was also added to each plate between samples to provide a barrier between cultures and to prevent evaporation. Virus cultures were passaged each week by transferring 100 μL of culture supernatant onto 0.5 million fresh CEM-GFP cells in 500 μL of CEM medium (RPMI 1640 medium supplemented with 10% Fetal Bovine Serum, 100 unit/mL penicillin, 100 µg/mL streptomycin, 2 mM l-glutamine, and 500 μg/mL G418). After inoculating the culture each week, the remaining supernatant was aliquoted and frozen at − 80 °C. The cell pellet was then resuspended in PBS and frozen at − 80 °C in preparation for genomic DNA (gDNA) isolation.
WST-1 cytotoxicity assay
The cytotoxicity was determined on CEM-GFP cells via a colorimetric assay to measure the relative proliferation rates of cells in culture. 7500 cells /well were seeded in 96-well plates and treated with varying concentrations of AAR029b and KR13 compounds at 37 °C. After 18 h of incubation, the media containing compounds were discarded and treated with 10% (v/v) WST-1 (a tetrazolium salt) in fresh media for one hour at 37 °C. The cell viability was determined by measuring the absorbance at 450 nm using a Tecan Infinite F50 plate reader.
Neutralization assay with a fully-infectious virus strain
TZM-bl cells were seeded into 96-well plates at a density of 10,000 cells /well. After 24 h growth, HIV-1 strain fully-infectious NL4-3 was incubated with serial dilutions of inhibitors (AAR029b, KR13 or 17b) for 30 min at 37 °C before adding the mixture to the cells. The virus was allowed to infect target cells for 24 h. Cells were lysed using 1X passive lysis buffer (Promega). Three freeze–thaw cycles were conducted to help with cell lysis. 40 μL of cell lysate was transferred to a white 96-well plate (Greiner), mixed with 100 μL luciferin buffer (0.1 M potassium phosphate, 0.1 M magnesium sulfate, 0.1 M ATP and DTT) and 50 μL 1 mM D-luciferin (Anaspec). Luminescence was immediately measured using a Wallac 1450 Microbeta Luminescence reader at a wavelength of 490 nm. The data were analyzed in GraphPad Prism 9 using nonlinear regression fit with dose response-inhibitor equation (4 parameters) shown as below:
$$Y = {\text{Bottom }} + \frac{{\left( {{\text{Top}} - {\text{Bottom}}} \right)}}{{\left( {1 + \left( \frac{X}{IC50} \right)^{HillSlope} } \right)}}$$
(1)
Top and Bottom are the response values at high and low inhibitor concentrations, respectively. X and Y represent as the inhibitor concentrations and infection levels, respectively. IC50 is the concentration of inhibitor, at which gives a response halfway between Top and Bottom. The Hill Slope describes the slope of the sigmoidal curve between the Top and Bottom plateaus.
Isolation and sequencing of the HIV-1 genome
Previously frozen cells for the desired week were taken out of the − 80 °C freezer and thawed. Genomic DNA from infected cells were obtained using the QIAamp DNA Mini and Blood Mini Kits (Qiagen) and following the manufacturer’s protocols. The HIV-1 Env genome was amplified using the specific five pairs of PCR primers. 1st: 5ʹ-CTATGGCAGGAAGAAGCGGAGAC-3ʹ and 5ʹ-ACCAGCCGGGGCACAATAATG-3ʹ; 2nd pair: 5ʹ-GCATGAGGATATAATCAGTTTA TGGG-3ʹ and 5ʹ-AATTCCCCTCCACAATTAAAACTG-3ʹ; 3rd pair: 5ʹ-GCTGTTAAATGGCA GTCTAGC-3ʹ and 5ʹ-CAAATGAGTTTTCCAGAGCAACCC-3ʹ; 4th pair: 5ʹ-GAGAAGAGTG GTGCAGAGAG-3ʹ and 5ʹ-CTATCTGTCCCCTCAGCTACTGC-3ʹ; 5th pair: 5ʹ-GATTGTGGA ACTTCTGGGAC-3ʹ and 5ʹ-TCCCAGGCTCAGATCTGGTCTAAC-3ʹ. Each primer set generated an approximately 1 kB fragment that overlapped with another PCR fragment to ensure full coverage of the Env genome. Amplification was accomplished using Phusion® High-Fidelity polymerase kit (New England Biolabs). The reaction for amplification was optimized as follows: 1 × High Fidelity buffer, 2.5 mM MgCl2, 0.35 mM dNTP mix, 0.3 mM of forward and reverse primers, and 0.5 units of Phusion® High-Fidelity polymerase. The reaction was initiated with 2 min of initial denaturation at 95 °C, followed by 40 cycles of amplification (94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min), and a final elongation step of 72 °C for 10 min. PCR products were confirmed by agarose gel running, purified by using Wizard® PCR Clean-Up kit (Promega), and sequenced using Sanger Sequencing service provided by Genewiz, LLC.
Site-directed mutagenesis of envelope expression plasmids
Site-directed mutagenesis were performed using a Quick-Change II XL Site-Directed Mutagenesis kit (Stratagene). Primers were designed and synthesized by IDT Inc. using solid-phase synthesis. Confirmed mutant plasmids were transformed into MAX Efficiency Stbl2 cells (Stratagene) to better support [23, 37,38,39] the retrovirus vector and produced in a large quantity using a Hispeed® Plasmid Maxiprep kit (Qiagen). Double mutations were prepared by conducting a second round of site-directed mutagenesis on the basis of the purified and confirmed DNA template. All the sequences of constructs were confirmed through Sanger sequencing done by Genewiz, LLC.
Production and purification of envelope-pseudotyped virus
Neutralization assays were conducted with a luciferase reporter assay by using the single-round infection of envelope-pseudotyped viruses following a standard protocol [15, 21,22,23]. 8 μg envelope deficient provirus construct (pNL4-3.luc.AM [47]) with 4 μg Env expression vector (pDOLHIVenv /pHxBc2 Env) were co-transfected via a polyethyleneimine (PEI) transfection reagent into HEK293T cells. Supernatants were collected 48 to 72 h after transfection to obtain pseudotyped viruses. The supernatant with viral particles was spun down to remove cell pellet, filtered through a 0.45 μm polyethersulfone (PES) membrane to remove debris, and concentrated to 1.5 mL by a 100 kDa cut-off spin concentrator (Amicon Ultra Ultracell, Millipore). Gradient purification was performed with a continuous 6–20% iodixanol gradient. The concentrated virus supernatant was loaded on top of the 10 mL gradient. After a two-hour ultracentrifuge spin at 40,000 RPM at 4 °C, 10 fractions were tested for the presence of virus and the fractions 6–8 with viruses were confirmed and then pooled together. 0.3 mL aliquots were stored in -80 °C after fast freezing by dry ice. Purified viruses were validated by ELISA and infectivity assay.
Neutralization assay with envelope-pseudotyped virus
HOS.T4.X4 cells were seeded at a density of 7500 cells per well in 96-well plates and cultured for 24 h at 37 °C. HIV-1 NL4-3 or HxBc2 pseudovirus corresponding to 500 ng p24 was incubated with serial dilutions of inhibitors (AAR029b, KR13 and 17b) for 30 min at 37 °C. The pseudovirus-inhibitor mixtures were loaded onto the seeded HOS.T4.X4 cells and incubated at 37 °C. Medium was changed after 24 h. Cell lysis was performed after another 24 h using 1X passive lysis buffer (Promega). Three freeze–thaw cycles were conducted to help with cell lysis. 40 μL of cell lysate was transferred to a white 96-well plate (Greiner), and mixed with 100 μL luciferin buffer (0.1 M potassium phosphate, 0.1 M magnesium sulfate, 0.1 M ATP and DTT) and 50 μL 1 mM D-luciferin (Anaspec). Luminescence was immediately measured using a Wallac 1450 Microbeta Luminescence reader at a wavelength of 490 nm. The data were analyzed in GraphPad Prism 9 using nonlinear regression fit with dose response-inhibitor equation (4 parameters).
Protein expression and purification
The plasmid for HxBc2 gp120 wild type and mutants in pcDNA3.1(−) vector for transient transfection was purified using a ZymoPURE II Plasmid Maxiprep kit (Zymo Research) and transfected into HEK293F cells according to manufacturer’s protocol (Invitrogen). After five days of inoculation at 37 °C, cells were harvested and spun down. The supernatant was filtered through 0.2 μm filters. Purification was conducted over a 17b antibody-coupled column prepared using an NHS-activated Sepharose, HiTrap HP column (GE Healthcare). gp120 subunits were eluted by using 0.1 M glycine buffer (0.15 M NaCl, pH 2.4). The eluted protein was rapidly neutralized by the addition of 1 M Tris buffer (pH 8.0). Then buffer exchange was performed with 1 × PBS pH 7.4. The protein samples were applied to a HiLoad 26/60 Superdex 200 HR prepacked gel filtration column (GE). Purity of eluted fractions and monomeric state of HxBc2 gp120 were identified by SDS-PAGE/immunoblotting with mAb 16H3 (which recognize the C1 region of gp120). Monomeric gp120 were pooled, concentrated, frozen and stored in aliquots at − 80 °C.
Thermodynamic profiles of binding by isothermal titration calorimetry (ITC) analysis
Equilibrium dissociation constants of AAR029b, KR13 and 17b IgG were determined at 25 °C on a VP-Isothermal Titration Calorimetry (VP-ITC) system (MicroCalTM, GE Healthcare, Freiburg). 60–100 µM of KR13, AAR029b or 17b in ITC Buffer (1 × PBS pH 7.4) were titrated in 8 µL steps into a reaction chamber containing 2–8 µM of HxBc2 gp120 WT, V255I and V255I/S143N monomers in the same buffer. The resulting heat changes upon injection were integrated over a time range of 240 s, and the obtained values were fit to a standard single site-binding model using Origin® software.
Surface plasmon resonance (SPR) interaction analysis
SPR experiments were performed on a Biacore 3000 biosensor (Global Cytiva Lifesciences) at 25 °C using PBS-P (10 mM Phosphate, 150 mM NaCl, pH 7.4, 0.005% P-20) as the running buffer. A CM5 sensor chip was docked and derivatized by amine coupling with HIV-1HxBc2 WT and mutant gp120 subunits using freshly prepared 1:1 50 mM NHS (N-hydroxysuccinamide):200 mM EDC (1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide). Flow cell 1 was left free to serve as a control for flow cells 2–4 containing 1800–2200 response units (RU) of HIV-1 HxBc2 gp120 WT, V255I and V255I/S143N monomers. Direct binding was determined by injecting AAR029b, KR13 or 17b IgG at concentrations spanning 0.20 nM to 3200 nM over all flow cells at a flow rate of 50 μL/min for 5 min (association phase) and all flow cell surfaces were washed with running buffer for 5 min (dissociation phase). The remaining bound analytes were removed with 1 pulse of 10 mM Glycine (pH 2.5) for 40 s. Next, soluble CD4 spanning 0.14 nM to 300 nM was injected over all flow cells at a flow rate of 50 μL/min with 5 min association phase and 10 min dissociation phase. Surface regeneration was attained using one pulse of 10 mM glycine (pH 2.5) for 40 s.
For data analysis, binding profiles were double referenced to minimize the impact of instrument and solvent noise. For AAR029b, 17b and CD4 interactions, the sensorgrams from two data sets were globally fit using Scrubber (BioLogic Software) to a Langmuir 1:1 binding model to obtain second-order association rate constant k1 and first-order dissociation rate constant k−1. The equilibrium dissociation constant was calculated as KD = k−1/k1. For KR13 interactions, the sensorgrams were fit to a three-state binding/isomerization model as depicted below:
$${\text{Env }}\begin{array}{*{20}c} {{\text{k}}_{1} \left[ {{\text{PT}}} \right]} \\ \rightleftharpoons \\ {{\text{k}}_{ - 1} } \\ \end{array} {\text{ Env}} \cdot {\text{PT }}\begin{array}{*{20}c} {{\text{k}}_{2} } \\ \rightleftharpoons \\ {{\text{k}}_{ - 2} } \\ \end{array} {\text{ Env}} \cdot {\text{PT}}^{*}$$
(2)
The model depicts a biomolecular interaction between KR13 (here depicted as PT) and Env followed by a hypothesized disulfide rearrangement induced by the inhibitor thiol. Rate constants k1 and k-1 are defined as they are in the 1:1 binding model, while k2 and k-2 are first order rate constants describing isomerization and de-isomerization, respectively. Equations describing the time course of the interactions can be found in Additional file 1: Method S1. For the fit, it was assumed that the states \(\mathrm{Env}\cdot \mathrm{PT}\) and \({\mathrm{Env}\cdot \mathrm{PT}}^{*}\) had the same SPR signal. Rate constants were determined using a least-squares gradient-step algorithm coded in Visual Basic and implemented through Microsoft Excel. The KR13 equilibrium dissociation constant was calculated as:
$$K_{D} = \frac{{k_{ - 1} k_{ - 2} }}{{k_{1} \left( {k_{2} + k_{ - 2} } \right)}}$$
(3)
Molecular modeling of the peptide triazole binding to a fully-glycosylated HIV-1 trimer
Preparation and molecular docking were performed using an established methodology [14]. Briefly, peptides (UM15 and AAR029b) and protein (chain C of the Env trimer PDB code 5FUU [9] were prepared for the docking study in silico by using The Schrödinger package (Schrödinger Suite 2014; Schrödinger, LLC) and by Autodock tools graphical interface (MGtools 1.5.6rc3). These minimized and repaired structures were saved as PDB files for use in the docking simulations. Flexible docking was carried out using Autodock and setting Trp112 and Trp427 as flexible. Cluster analysis was performed on docked results, with a root-mean-square tolerance of 2.0 Å. Visual inspection of the lower energy docked poses was compared to the prior modeling work with the monomeric gp120 [15]. Considering the mutagenesis analysis results, the lowest binding energy pose that matched the mutational and structure–activity analyses [15] was selected as a representative complex for each compound. Molecular graphics and steric clash analyses were performed with the UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311) [48].
Determination of CD4 utilization in viral entry
Neutralization assay was performed as described previously by inhibiting HIV-1 recombinant virus NL4-3 infection with D23.2 (DARPin 23.2 [36]). The infectivity levels of HIV-1NL4-3 WT and mutants (S143N, V255I, V255I/S143N) were determined in the presence of D23.2 (11 nM, about IC50 value). We displayed the absolute infectivity in the presence and absence of D23.2. The relative infectivity (+D23.2/−D23.2) was plotted to better indicate the CD4 utilization in the WT and mutant recombinant viruses.
gp120 shedding analysis
The gp120 shedding assay was performed as described previously [49]. Briefly, three million HEK293T cells per flask were transiently transfected with 4 μg of HIV-1 NL4-3 Env plasmid and transfection reagent FuGENE 6 (Promega). 5 mM EDTA in DPBS was used to detach cells at 24 h and cells were reseeded in 96-well plates with 50,000 cells per well. Mock and transfected HEK293T were treated with serial dilutions of sCD4 183, AAR029b or a PBS control for 2 h at 37 °C. Cells were washed with fresh medium, detached with 5 mM EDTA, and then washed and resuspended in FC buffer (1% BSA and 1 mM EDTA in 10 mM PBS pH 7.4). 2% paraformaldehyde in DPBS was used to fix the cells for 15 min at room temperature. Cells were then washed and resuspended in the FC buffer three times. The fixed cells were stained with 5 μg/mL antibody 35O22 (NIH AIDS Reagent Program) for one hour at room temperature and washed three times with FC buffer. Secondary stain anti-Human PerCP in 1:500 dilution (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was incubated with cells for one hour at room temperature, and washed three times with FC buffer. Cell counts and Env staining were assessed in counted cells on a Guava EasyCyte 5HT flow cytometry system (Millipore). Analyzed cells were subjected to forward/side scatter gating based on untreated control populations of identical transfection, and median fluorescence values were obtained using Guava InCyte 3.2 software. Data were analyzed in GraphPad Prism 9 using nonlinear regression fit with dose response-inhibitor equation (4 parameters).