PAV-620, and PAV-805 are chemical analogs from a protein assembly modulating small molecule lead series that displays broad anti-cancer activity. These compounds are active against all cancers in the NCI-60 cancer cell line screen, which is representative of diverse blood, brain, breast, colon, lung, ovarian, prostate, renal, and skin cancers. Safety in mice is observed up to doses of 10 mg/kg daily and nontoxicity to healthy human peripheral blood mononuclear cells at doses up to 20uM. The compounds are as efficacious as Paclitaxel in reducing tumor growth and metastasis in the aggressive 4T1 mouse allograft model for triple negative breast cancer. The mechanism of action of PAV-620 and PAV-805 in primary carcinoma cells under conditions inducing programmed cell death was found to be distinct from the cytotoxic mechanisms of Staurosporine, Paclitaxel, and Etoposide. PAV-805 was shown to target a subset of protein disulfide isomerase in a dynamic multi-protein complex enriched in the cancer hallmark proteins involved in reprogramming energy metabolism. These data suggest a new approach to cancer therapeutics, with selectivity arising not from targeting PDI itself, an abundant cellular protein, but from targeting a disease-associated assembly state of PDI-containing complexes as a therapeutically exploitable layer of cancer biology.

National Cancer Institute 60 Cell Line Screen

In the NCI-60 screen, up to 60 cancer cell lines (CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226, SR, A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522, COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620, SF-268, SF-295, SF-539, SNB-19, SNB-75, U251, LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI/ADR-RES, SK-OV-3, 786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31, PC-3, DU-145, MCF7, MDA-MB-231/ATCC, HS 578T, BT-549, T-47D, MDA-MB-468) are grown.

Two methods have been used to assess anti-tumor cytotoxicity in the NCI-60 screen.

In the Classic Screen (method used through September 2023), cells were grown for 24 hours then treated with vehicle or compound, or fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition. The treated cells were grown for an additional 48 hours before being fixed in situ with TCA. Fixed cells were then stained with Sulforhodamine B. Absorbance was read to determine cell viability of compound-treated cells relative to both the time at which treatment began and to untreated cells at the end of the study.  The NCI-60 screen was initially conducted at a single dose of 2.5uM, but was subsequently repeated with a dose titration.

The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 μL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37° C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of experimental drugs.

After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/ml gentamicin

Following drug addition, the plates are incubated for an additional 48 h at 37°C, 5 % CO2, 95 % air, and 100 % relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 μl of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant is discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4 % (w/v) in 1 % acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is removed by washing five times with 1 % acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80 % TCA (final concentration, 16 % TCA).

In the HTS384 Screen (method used after September 2023), the sixty human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. To initiate a screen, 40 μl of cells are inoculated into the wells of white 384-well microtiter plates at plating densities ranging from 250 to 2,500 cells/well, depending on the doubling time of individual cell lines. After inoculation, the microtiter plates are incubated at 37°C with 5% CO² and 95% relative humidity for 24 h before the addition of controls and test agents. Prior to a screen, the test agents and controls are solubilized in dimethyl sulfoxide (DMSO) at 400-fold the final test concentration and stored frozen in polypropylene 384-well microtiter plates.

Twenty-four hours after cell line inoculation, acoustic dispensing is used to transfer 100 nl of DMSO (0.25% (v/v), final) into the wells of microtiter plates, each containing a single cell line. Subsequently, 40 μl of CellTiter-Glo are dispensed into the wells, according to the manufacturer's protocol. Luminescence is measured to assess cell viability at the time of drug addition (time zero, Tz). Acoustic dispensing is also used to transfer 100 nl of controls and test agents (400-fold dilution, final) into duplicate microtiter plates for each cell line to achieve technical replicates. Controls in each microtiter plate include vehicle, 100% cytotoxicity (1 μM Staurosporin NSC755774 and 3 μM Gemcitabine NSC613327 [n = 8]), and five concentrations of doxorubicin (NSC123127, 25 μM [n = 2], 2.5 μM [n = 1], 250 nM [n = 2], 25 nM [n = 1], 2.5 nM [n = 2]. Following the delivery of controls and test agents, the microtiter plates are incubated for 72 h at 37°C with 5 % CO² and 95% relative humidity. After 72 h of exposure, 40 μl of CellTiter-Glo are dispensed into the wells of the microtiter plates and luminescence is measured, according to the manufacturer's protocol, to assess cell viability. 

For both methods, using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug concentrations levels. Percentage growth is calculated as:

[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz

[(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz.

Three dose response parameters are calculated for each experimental agent. Growth inhibition of 50 % (GI50) is calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) is calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment is calculated from [(Ti-Tz)/Tz] x 100 = -50.

PAV-951, PAV-0383, PAV-0384 were assessed in the Classic Screen. All others were assessed by the HTS384 screen.

Cytotoxicity Assessment on PBMCs and H9 Lymphoma

Healthy human PBMCs and H9 cells were seeded at a density of 5,000 cells/well and treated with either DMSO or compound, at concentrations of 2.5uM, 5uM, 10uM, and 20uM. The cells were incubated at 37oC for 72 hours then cell viability was measured using an AlamarBlue^TM assay.

Anoikis Assay

BB5 and BB7 cells were seeded at 2000 cells/well into PhenoPlate 384-well microplates (Cat # 6057302, Revvity) pre-treated with PolyHEMA coating. Briefly, 20ul of 20mg/mL PolyHEMA was added to each well, then allowed to air dry with the lid open in a biosafety cabinet to ensure sterility. After drying, the same amount was added to the wells a second time. Once dried, the plate was used for anoikis assays. Drug compounds were added along with seeded BB5 and BB7 cells. Live cell stains ChromaLIVE non-toxic dye (Saguaro Biosciences) and Annexin-V labelled with Alexa-647 (made in house) were added to the cells upon seeding. Each concentration had a duplicate as a technical replicate. DMSO conditions had 6 technical replicates. At 24 hours post-treatment, Hoechst 33342 at 1µg/mL was added and the cells were imaged on a Revvity Opera Phenix screening confocal microscope. A total of 16 fields-of-views were imaged for each well.  Harmony image analysis software was used to post-process the images. A total of 293 features related to intensity, morphology, and texture features were extracted. A random forest model was generated in python from the package sklearn, using sampled images of cells (from both BB5 and BB7 cell types) from the DMSO condition as ‘DMSO’, and 2.5µM Staurosporine as ‘Dead’. Normalized area under the curve was calculated as the total AUC after the baseline death percentage (percent of dead cells at 0µM) was removed from all data points.

Warburg Assay

Glucose concentrations in cell culture supernatants were determined using the glucose assay kit (Abcam, #65333), in accordance with the manufacturer’s protocols. MCF7 cells (2 × 10⁴ per well) were seeded in 96-well plates. After 24 h, the medium was replaced with phenol red–free DMEM supplemented with 5% dialyzed fetal bovine serum (FBS) and 1.1 mM glucose. Cells were treated with the indicated compounds and incubated for 20 h. Supernatants were subsequently collected and directly analyzed for glucose content. An outlier in the PAV-805-treated sample was excluded from the analysis.  Cell viability of cells treated with compound under matched conditions was evaluated using a resazurin (Alamar Blue) assay. Briefly, 10 µL of a 100 µg/mL resazurin solution was added to each well, followed by incubation for 2 h at 37 °C. Absorbance was measured at 570 nm with a reference wavelength of 620 nm. The assay was repeated three times.

Mouse Safety

For the intraperitoneal MTD study, female Balb/c mice aged 8-10 weeks were randomly divided into treatment groups with three animals per group. Animals in each treatment group were weighed and received one IP injection of 0.1-0.15mL containing either vehicle (10% DMSO, 45% propylene glycol, 45% sterile water), or varying doses compound. Animals were observed from day 0 until day 3 for clinical signs of toxicity. Animals were euthanized after 72 hours and were examined externally and internally by a pathologist for abnormalities in organ weight and tissue damage. Blood samples were sent for a complete blood count bioanalysis. MTD was determined to be the dose at which no signs of toxicity were observed by any parameters.

Mouse Allograft

A suspension of 0.1x10⁶ cells were implanted into the mammary fat pad of normal balb/c mice. Size of the tumor was measured using a digital Vernier caliper. Tumor volume was calculated as the  [Length (L) × Width (W) ^2^] /2, where length (L) is the largest diameter of the tumor and width (W) is the smallest diameter. Animals were treated by IP injection daily with either vehicle (N=6), 2 mg/kg Paclitaxel (N=6), 5 mg/kg then (after 8 days) 1.5 mg/kg PAV-620, or 10mg/kg PAV-0805 (N=6), beginning when the tumor volume reached 50mm³. An additional late treatment group (N=3) received vehicle until the tumor volume reached 500mm³, and were subsequently treated with 10mg/kg PAV-0805 daily. Tumor growth inhibition was calculated based on the following formula:

% TGI = {(TV_vehicle Final - TV_vehicle Initial) - (TV_treatment Final - TV_treatment Initial)} x 100                                                                  (TV_vehicle Final - TV_vehicle Initial)

Animals were sacrificed once the tumor in the vehicle-treated group reached a volume of 2000mm³. Lung tissues were isolated and visually observed for metastatic cancer cell colonies.

Drug Resin Affinity Chromatography

A549, HT29, or U251 cells were grown in minimum essential media (UCSF) with 10% FBS and 1% Penstrep for 48 hours. In some conditions cells were treated with PAV-0805 for 20 hours. Cells were scraped into cold phosphate buffered saline (PBS) (10mM sodium phosphate, 150 mM sodium chloride pH 7.4), then spun at 1,000 rpm for 10 minutes until pelleted. The PBS was decanted and the pellet resuspended in a low salt buffer (10mM HEPES pH 7.6, 10mM NaCl, 1mM MgAc with 0.35% Tritonx100) then centrifuged at 10,000 rpm for 10 minutes at 4o C. The post-mitochondrial supernatant was removed and adjusted to a concentration of approximately 5 mg/ml unless otherwise stated and equilibrated in a physiologic column buffer (50 mM Hepes ph 7.6, 100 mM KAc, 6 mM MgAc, 1 mM EDTA, 4mM TGA). The extract was supplemented with an energy cocktail (to a final concentration of 1mM rATP, 1mM rGTP, 1mM rCTP, 1mM rUTP, creatine phosphate and 5 ug/mL creatine kinase). 30 ul of extract was then incubated for one hour at 22oC on 30 ul of affi-gel resin coupled to PAV-951, PAV-0805, or a 4% agarose matrix (control). The input material was collected and the resin was then washed with 3 ml column buffer to remove any nonspecifically bound material. The resins were eluted overnight at 22oC in 100ul column buffer containing 100uM PAV-951 or PAV-0805 with the energy cocktail. Eluate samples were sent for MSMS analysis.

Chemical Photocrosslinking

Cell extract was prepared as above then adjusted to a protein concentration of approximately 2 mg/ml in column buffer (50 mM Hepes pH 7.6, 100 mM KAc, 6 mM MgAc, 1 mM EDTA, 4mM TGA). 70ul of 2uM stock of photocrosslinker analogs of PAV-805 chemically modified to contain biotin and a diazirine group or a control containing the biotin and diazarine but not assembly modulator compound prepared in column buffer were added to 70ul of extract, incubated for one hour at on ice, then exposed to ultraviolet light for 10 minutes on ice. After crosslinking, samples were divided in two 60 ul aliquots and one set was denatured by adding 6ul of 10% SDS, 0.75 ul DTT, and boiling for 10 minutes. Both native and denatured aliquots were then diluted in 1200 ul column buffer containing 0.1% triton. 5 ul of magnetic streptavidin beads were added to all samples and mixed for one hour at room temperature to capture all biotinylated proteins and co-associated proteins.  Samples were placed on a magnetic rack to hold the beads in placed and washed three times with 1mL of column buffer containing 0.1% triton. After washing, beads were resuspended in 40 ul of gel loading buffer containing SDS and analyzed by western blot for PDI and KAP1.
 

Silver Stain

SDS/PAGE gels were incubated overnight in a fixative (50% methanol, 10% acetic acid, 40% water), then for an hour in 50% methanol (done as two washes), and an hour in water (done as two washes). The gels were sensitized in 0.02% sodium thiosulfate for one minute then washed twice for 30 seconds with water. The gels were incubated for 30 minutes in cold 0.1% silver nitrate with 0.02% formaldehyde then washed twice for 30 seconds. The gels were developed in 3% sodium carbonate with 0.02% formaldehyde. The developed gels showing the pattern of protein bands was scanned and the image was analyzed.

Western blotting

SDS/PAGE gels were transferred in Towbin buffer (25mM Tris, 192mM glycine, 20% w/v methanol) to polyvinylidene fluoride membrane, blocked in 1% bovine serum albumin (BSA) in PBS, incubated overnight at 4 degrees Celsius in a 1:1,000 dilution of 100ug/mL affinity-purified primary IGG to ALDH1A1, GSTP1, PDI, PSMB5, KAP1, MDH, YWHAZ in 1% BSA in PBS containing 0.1% Tween-20 (PBST). Membranes were then washed twice in PBST and incubated for two hours at room temperature in a 1:5000 dilution of secondary anti-rabbit or anti-mouse antibody coupled to alkaline phosphatase in PBST. Membranes were washed two more times in PBST then incubated in a developer solution prepared from 100 uL of 7.5 mg/mL 5-bromo-4-chloro-3-indolyl phosphate dissolved in 60% dimethyl formamide (DMF) in water and 100ul of 15 mg/ml nitro blue tetrazolium dissolved in 70% DMF in water, adjusted to 50mL with 0.1 Tris (pH 9.5) and 0.1 mM magnesium chloride. Membranes were scanned and the integrated density of protein band was measured on ImageJ. Averages and the standard deviation between repeated experiments were calculated and plotted on Microsoft Excel.

MSMS Analysis

Samples were processed by SDS PAGE using a 10% Bis-ttris NuPAGE gel with the MES buffer system. The mobility region was excised and washed with 25 mM ammonium bicarbonate followed by 15mM acetonitrile. Samples were reduced with 10 mM dithoithreitol and 60 degrees Celsius followed by alkylation with 5o mM iodacetamide at room temperature. Samples were then digested with trypsin (Promega) overnight (18 hours) at 37 oC then quenched with formic acid and desalted using an Empore SD plate. Half of each digested sample was analyzed by LC-MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Peptides were loaded on a trapping column and eluted over a 75uM analytical column at 350 nL/min packed with Luna C18 resin (Phenomenex). The mass spectrometer was operated in a data dependent mode, with the Oribtrap operating at 60,000 FWHM and 15,000 FWHM for MS and MS/MS respectively. The fifteen most abundant ions were selected for MS/MS.

Data was searched using a local copy of Mascot (Matrix Science) with the following parameters: Enzyme: Trypzin/P; Database: SwissProt Human (concatenated forward and reverse plus common contaminants); Fixed modification: Carbamidomethyl (C) Variable modifications: Oxidation (M), Acetyl (N-term), Pyro-Glu (N-term Q), Deamidation (N/Q) Mass values: Monoisotopic; Peptide Mass Tolerance: 10 ppm; Fragment Mass Tolerance: 0.02 Da; Max Missed Cleavages: 2. The data was analyzed by label free quantitation (LFQ) and spectral count methods. LFQ intensity values of each condition were measured in triplicate and compared against each other to generate log2 fold change values for each combination of conditions. Spectral counts were filtered for a 1% protein/peptide false discovery rate requiring 2 unique peptides per protein.

The data set was further adjusted by subtraction of spectral counts for specific proteins observed in the control resin, and subtraction of proteins that were not found in all triplicate-repeat samples. The adjusted dataset was searched for enrichment of cancer hallmarks in the cancerhallmarks.com dataset as described in https://doi.org/10.1016/j.jpha.2024.101065.

Molecular Modeling

Docking studies were performed using ICM Pro (Molsoft LLC)(84). A human protein disulfide isomerase (PDI) model was prepared from the crystal structure (PDB ID: 3F8U)(44). The 3F8U file was imported into ICM, converted to an ICM object, and energy-minimized using default program settings. Putative binding pockets were then identified using the ICM pocket-finding algorithm with standard search parameters. PAV-805 and PAV-951 were subsequently docked into the selected pocket(s) according to the ICM docking workflow. Docking was conducted with a thoroughness setting of 50 (effort per ligand), generating three conformations per ligand. Docked poses were evaluated using the resulting Mean Force scores (mf scores) -  the potential of mf score evaluates the strength of interactions based on statistical preferences of atom-atom contacts. The lower mf score, i.e. more negative score represents a better, more energetically stable predicted binding pose.