Two structurally-unrelated small molecule chemotypes, represented by compounds PAV-617 and PAV-951 with antiviral activity in cell culture against Mpox virus (Formerly known as monkeypox virus) and human immunodeficiency virus (HIV) respectively, were studied for anti-cancer efficacy. Each exhibited apparent pan-cancer cytotoxicity, reasonable pharmacokinetics, and non-toxicity in mice at active concentrations. Anti-tumor properties of both chemotypes were validated in mouse xenografts against A549 human lung cancer and, for one of the chemotypes against HT-29 colorectal cancer. The targets of these compounds are unconventional: each binds to a different transient, energy-dependent multi-protein complex. Treatment with these compounds alters the target multi-protein complexes in a manner that appears to remove a block, crucial for cancer survival and progression, on a homeostatic linkage between uncontrolled proliferation and apoptosis. These compounds provide starting points for development of novel, next-generation, non-toxic, pan-cancer therapeutics.

Chemical Synthesis

All compounds synthesized were confirmed by LCMS with purity typically > 98%

Synthesis of PAV-617 (see Supplemental Figure 1A)

Phenothiazin-5-ium tetraiodide hydrate

Phenothiazin-5-ium tetraiodide hydrate:  a solution of phenothiazine (4.98 g, 25 mmol) in anhydrous chloroform (50 mL) was stirred at 50C and the solution of iodine (12.7 g, 50 mmol) in CHCl3 (250 mL) was added dropwise over 4h. The resulting dark solution was stirred for an additional 3h at 50C, monitored by TLC. After the disappearance of the starting material, the resulting precipitate was filtered, washed with a copious amount of chloroform, dried overnight in vacuo to afford a dark solid (13.9 g, 74%).

3,7-Di(pyrrolidin-1-yl) phenothiazinium iodide

A solution of phenothiazin-5-ium tetraiodide hydrate (2.8 g, 3.6 mmol) in mixture acetonitrile/methanol (50 mL) and pyrrolidine (710 mg, 10 mmol) was stirred for    4 h at room temperature. The resulting mixture was concentrated to dryness and purified by flash chromatography using the methanol-chloroform gradient to purify the desired compound.

Synthesis of PAV-617 resin (see Supplemental Figure 1B)

2-Aminomethylphenothiazine

2-Cyanophenothiazine (2.24 g, 10 mmol) was dissolved in THF (50 mM). Solution of 1 M lithium aluminum hydride (LiAlH4) in THF (20 mL, 40 mmol) was added at room temperature and reaction mixture was stirred for 24 hours. Mixture was diluted with ether and cooled to 00C To this solution water (1.5 mL) (slowly), 15% aqueous sodium hydroxide (1.5 mL) and water (4.5 mL) were added. Mixture was warmed to RT and stirred 15 min. Some anhydrous MgSO4 was added, stirred 15 min., filtered to remove salt. Solution was concentrated and product was used without purification. Yield – 1.9 g (83% theor). M+1 = 229 (LCMS)

2-Bocaminomethylphenothiazine

2-Aminomethylphenothiazine (4.0 g, 17.5 mmol) was dissolved in DCM (100 mL) and Boc-anhydride (3.85 g, 17 .5 mmol) was added with stirring at RT. After 1 h mixture was concentrated and product was purified with flash chromatography (solvent system: hexane – ethyl acetate). Yield – 4.0 g (70 % theor.). M+1 = 329

2-Bocaminomethylphenothiazin-5-ium tetraiodide hydrate

Solution of 2-Bocaminophenothiazine (984 mg, 3 mmol) in anhydrous chloroform (10 mL) was stirred at 50 C and the solution of iodine (3.81 g, 15 mmol) in CHCl3 (90 mL) was added dropwise over 4h. The resulting dark solution was stirred for an additional 3h at 50 C, monitored by TLC. After the disappearance of the starting material, the resulting precipitate was filtered, washed with a copious amount of chloroform, dried overnight in vacuo to afford a dark solid. Yield – 1.5 g (60% theor.).

2-Bocaminomethyl-3,7-di(pyrrolidin-1-yl) phenothiazin-5-ium iodide

A solution of 2-Bocaminomethylphenothiazin-5-ium tetraiodide hydrate (426 mg, 0.5 mmol) in chloroform (10 mL) and pyrrolidine (85 mg, 0.1 mL, 1.2 mmol) was stirred for 1 h at room temperature. The resulting mixture was concentrated to dryness and purified by flash chromatography using the methanol-chloroform gradient to provide the title compound. Yield – 207 mg (70% theor.) M = 465 (LCMS)

2-Aminomethyl-3,7-di(pyrrolidin-1-yl) phenothiazin-5-ium dihydrochloride

2-Bocaminomethyl-3,7-di(pyrrolidin-1-yl) phenothiazin-5-ium iodide (590 mg, 1 mmol) was mixed with 4M solution of HCl in 1,4 dioxane (5 mL). Solution was stirred 3 h at room temperature. The resulting mixture was concentrated to dryness and purified by flash chromatography using the methanol-chloroform gradient to provide the title compound. Yield – 280 mg (70% theor.) M+1 = 365 (LCMS)

To a solution of affi-Gel (Bio-Rad, 10 mL) in a solid phase synthesis tube with frit was added a solution of 2-Bocaminomethyl-3,7-di(pyrrolidin-1-yl) phenothiazine-5-ium iodide (54 mg, 0.11 mmol) and DIEA (1.0 mL) in isopropyl alcohol (4 mL) and the tube was put in a shaker for 12 h. Excess reagents were drained and the resin was washed with isopropyl alcohol (3×) and then saved in isopropyl alcohol.

Synthesis of PAV-951 (see Supplemental Figure 2A)

A mixture of 4,7-Dichloroquinazoline [(0.811g (4.1 mmol)] 1 and Isopentylamine [1g (12 mmol)] in 5mL of ACN was briefly sonicated and then stirred at room temperature for 20min and then diluted with EtOAc. The EtOAc solution was then washed with water, dried (Na2CO4) and the solvent removed. Crude material was columned with ISCO EtOAc/ CHCl3 affording 1g (97%) of 2.

To a mixture of 7-chloro-N-isopentyl-quinazolin-4-amine  [1g (4mmol)] 2, RuPhosPd [150mg (0.2mmol)] , RuPhos 90mg (0.2mmol), Cs2CO3  [3.93g (10 mmol)]  and 20mL of t-BuOH was added piperazine [172mg (2mmol)] . The mixture was heated and stirred at 870C for 20h. After cooling to room temp the mix was diluted with 20mL of sat. NaCl solution and extracted 2X with EtOAc. The extracts were dried (Na2SO4) and the solvent removed. The crude was purified with ISCO using 10%NH3 in MeOH / CHCl3 affording 600mg (60%) of the dimer PAV-951 (3).

Synthesis of PAV-951 resin (see Supplemental Figure 2B)

A mixture BocPiperazine [223mg(1.2mmol)], Cs2CO3 [600mg (1.5mmol)], PdRuPhos [37mg (0.05mmol)], RuPhos [23mg (0.05mmol)] and the 7-chloro-N-isopentyl-quinazolin-4-amine [250mg (1mmol)] 2 in 2-3mL t-BuOH was heated to 870C with stirring under argon for 20h. After cooling the mixture was diluted with 20mL of 10%MeOH/DCM and the resulting mix was filtered through a short pad of celite. The filtrate was rotary evaporated to dryness and the residue was purified on the Isco (Hexane / EtOAc) affording 384mg (96%) of the Bocpiperazine adduct 4.

A mixture of 4,7-Dichloroquinazoline [(0.189g (0.95 mmol)] 1 and tert-Butyl 4-(2-aminoethyl)piperidine-1-carboxylate [0.433g (1.9 mmol)] in 3mL of ACN was briefly sonicated and then stirred at room temperature for 20min and then diluted with EtOAc. The EtOAc solution was then washed with water, dried (Na2CO4) and the solvent removed. Crude material was columned with ISCO EtOAc/ CHCl3 affording 296mg (80%) of 5.

To Bocpiperazine adduct [223mg(1.2mmol)] 4 was added 3mL of 30% TFA / DCM and the mixture stirred for 1h at room temperature. The mixture was then rotary evaporated to dryness and the residue was partition between 1N NaOH and DCM. The DCM layer was dried (Na2SO4) and the solvent removed affording 228mg of the Piperazine adduct 6 which was used as is in the next step.

A mixture the piperazine compound [228mg(0.76mmol)], Cs2CO3 [376mg (1.15mmol)], PdRuPhos [33mg (0.04mmol)], RuPhos [19mg (0.04mmol)] and the Chloroquinazoline compound [296mg (0.76mmol)] 5 in 3mL t-BuOH was heated with stirring at 870C under argon for 20h. After cooling the mixture was diluted with 20mL of 10%MeOH/DCM and the resulting mix was filtered through a short pad of celite. The filtrate was rotary evaporated to dryness and the residue was purified on the Isco 10% NH3 in MeOH/CHCl3 affording 375mg (75%) of the desired unsymmetric dimer PAV-504 (7).

To the Dimer 7 [6.5mg (0.01mmol)] was added 0.5mL of 4N HCl in Dioxane and the resulting mix was allowed to stir for 30min at room temperature. The mixture was rotary evaporated to dryness and the residue dissolved in 1.5mL of iso-Propanol. Next DIEA [10ul (0.06mmol)] and a catalytic amount of DMAP [0.2mg] was added to the iso-Propanol solution. The resulting mix was added to affi-Gel-10 [1mL (0.015mmol)] in a fritted tube and the mixture was agitated on a shaker at room temperature overnight. Excess reagents were then drained and the resin was washed with 3mL (3x) of iso-Propanol and then stored in iso-Propanol affording 1mL of PAV-951 resin (8).

Experimental Models and Subject Details

Animal models

Maximum tolerated dose (MTD) studies were conducted using female Balb/c mice, aged 8-10 weeks or female CD1 mice, aged 5-6 weeks. Treatment groups were made up of 3 animals each, unless otherwise noted, and dosing regimens for disclosed data is provided. Animals were sacrificed at the end of the study period using an overdose of CO2. MTD studies were conducted at Vipragen Biosciences Private Limited or Radiant Research Services Private Limited in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Pharmacokinetic (PK) studies were conducted in male Sprague Dawley Rats aged 8-10 weeks or male CD1 mice aged 5-6 weeks or Balb/c nude mice. Treatment groups were made up of 4 animals each and dosing regimens for disclosed data is provided. Animals were sacrificed at the end of the study period using an overdose of CO₂. PK studies were conducted at Vipragen Biosciences Private Limited or Pharmaron in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Tumor xenograft studies were conducted using female Athymic Nude mice, strain CrTac: Ncr-Foxn1nu, aged 6-8 weeks. Tumor transplantation occurred through subcutaneous injection of a 0.1mL cell suspension containing 1 to 5x10⁶ A549 lung cancer cells obtained from ATCC in Matrigel in PBS into the left flank region of the mice. Treatment groups were made up of 6 animals each and dosing regimens for disclosed data is provided. Animals were sacrificed at the end of the study period using an overdose of isoflurane anesthesia. Both xenograft studies were conducted at Anthem Biosciences Private Limited in Bangalore, India and were approved by the Institutional Animal Ethics committee (IAEC) of Anthem Biosciences in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines and Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines.

Cell lines

The in vivo xenograft study utilized A549 (male human lung cancer) and HT-29 (female human colon cancer) cell lines. Cells were grown under sterile conditions. These studies were conducted at Anthem Biosciences Private Limited in Bangalore, India.

The human tumor cell proliferation assay used A172 (male human glioma), BFTC-905 (female human urinary bladder transitional cell carcinoma), COR-L105 (male human lung adenocarcinoma), DB (male human b-cell lymphoma), FaDu (male human pharynx squamous cell carcinoma), H9 (male human t-cell lymphoma), Hs 294T (male human melanoma), MCF7 (female human breast cancer), MDA MB 436 (female human breast cancer), MeWo (male human melanoma), MHH-PREB-1 (male human b-cell lymphoma), SJSA1-OSA (male human osteosarcoma), SU-DHL-10 (male human b-cell lymphoma), SW1353 (female human chondrosarcoma), and U-2 OS (female human osteosarcoma) cell lines. Two non cancer cell lines HMEC (breast) and HUVEC (umbilical vein) were assessed by the same method. This study was conducted by Eurofins Scientific as part of their OncoPanelTM.

The Mpox virus infectious virus assay used BSC-40 cells (nonhuman primate kidney) and Mpox Zaire 79 strain. These studies were conducted by the United States Army Medical Research Institute of Infectious Diseases in Frederick, Maryland.

Human immunodeficiency virus infectious virus assay used MT-2 cells (female human t-cell leukemia) and the NL4-3 Rluc reporter virus. These studies were conducted at the University of Washington in Seattle, Washington.

The in vitro screens for apoptosis, high-density/ low-density activity, and cell growth recovery utilized the A549, PANC-1, LNCaP C-33 (male human prostate cancer), LNCaP C-81 (male human prostate cancer), CHO-K1 (Chinese Hamster ovary), and Hennes 20 (Chinese Hamster ovary) cell lines. Cells were grown under sterile conditions. These studies were conducted at Prosetta Biosciences in San Francisco, California.

The in vitro drug resin affinity chromatography and photo-crosslinking experiments utilized A549 (male human lung cancer) cell line. Cells were grown under sterile conditions. Sterile conditions were not maintained once cells were harvested for in vitro experiments. These studies were conducted at Prosetta Biosciences in San Francisco, California.

BB1 and BB5 Adenoma versus Carcinoma screen

Two cell lines, BB1 and BB5, were derived from two patient breast biopsies that were obtained prior to treatment. Follow up classification indicated that BB1 was a benign adenoma and BB5 was a breast adenocarcinoma. The cells were grown in conditional reprogramming media in co-culture with irradiated fibroblasts as described in Liu et al., 2017(69). Once cultures reached exponential growth phase, the cells were harvested and treated with either PAV-617 or PAV-951. These studies were conducted at the Sunnybrook Health Science Center in Toronto, Canada.

National Cancer Institute Sixty Cell Line Screen

A panel of 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, PC-3, DU-145, MCF7, MDA-MB-231/ATCC, HS 578T, BT-549, T-47D, MDA-MB-468) were used to assess PAV-617, PAV-951, PAV-621, PAV-541, and PAV-436. This study was conducted at the National Cancer Institute.

In vitro experiments

Drug resin affinity chromatography and photo-crosslinking experiments, and SDS-PAGE/ silver stain/Western Blot analysis of the results, were conducted by Prosetta Biosciences in San Francisco, California under conditions described in figure legends. Results from disclosed in vitro experiments were repeated in triplicate unless otherwise stated. Mass spectrometry analysis of samples were conducted by MS Bioworks in Ann Arbor, Michigan.

Method and Analysis Details

Mpox virus infection assay

BSC-40 cells of 95% confluence in 24-well plates were infected with100 pfu of Mpox Zaire 79 diluted in Eagle’s Minimum Essential Medium with 2% fetal bovine serum and incubated in 37 degrees Celsius in 5% CO2 for 1 hour. The viral inocula were removed and replaced with the test compounds in six half log dilutions (0.1 mL per well) and the cells were overlaid with 1% methylcellulose in growth media (1 mL per well). The media and virus control cells received growth medium containing 1% methylcellulose. After three days of infection, when plaques appeared, cells were stained with crystal violet for an hour and then washed with water and dried overnight. The plaques were counted the next day and virus-only wells were compared with the compound-added wells to determine percentage protection. Infected cells were stained with crystal violet and viral plaques were counted. Averages and standard deviation for plaques observed under different treatment conditions were calculated in Microsoft Excel and graphed as the percent inhibition in PAV-617 treated cells compared to untreated cells.

Human immunodeficiency virus infectious virus assay

MT-2 cells were pre-seeded in 96-well plates in 100 ul of complete RPMI. Multiple concentrations of PAV-951 were serially diluted in DMSO then into an infection media prepared by diluting NL4-3 Rluc virus stock to 400 IU/100 ul with complete RPMI, which was transferred onto the MT-2 cells with a final MOI of 0.02 and final DMSO concentration of 1% in infected places. One well received DMSO only, instead of PAV-951, and one well received medium only for normalization and background collection. Cells were incubated at 37 degrees Celsius for 96 hours. 100ul of medium was removed and discarded and 10 ul of 15 uM EnduRen luciferase substrate was added to each well, followed by incubation for 1.5 hours at 37 degrees Celsius. Plates were read on a luminescence plate reader. Bioluminescence intensity was read on a Synergy H1 BioTek plate reader. Averages and standard deviation for viral titer observed under different treatment conditions were calculated in Microsoft Excel and graphed as the percent inhibition in PAV-951 treated cells compared to untreated cells.

Apoptosis Screen

A 96 well plate was seeded with Hennes 20 cells at 500 cells per well, CHO-K1 cells at 500 cells per well, LNCaP C-33 cells at 2000 cells per well, and LNCaP C-81 cells at 2000 cells per well. Cells were grown in 100uL minimum essential media for three days then three wells of each cell line received treatment with 1% DMSO. 12 hours after drug treatment, a mixture of 25 ul media and 25 ul Apo-ONE reagent (Promega) was added then the plate was covered and placed on a shaker at room temperature for six hours. The plate was read on a microplate reader for fluorescence at 499/521. Values were averaged and standard deviations were calculated for each triplicate condition and graphed on Microsoft Excel.

High density/ low density assay

Two 96 well plates were seeded with Hennes 20 cells in parallel where one was plated at a density of 500 cells/well and the other was plated at a density of 15,000 cells/well. 90 ul of minimum essential media was added to each well and plates were placed in a 37 degrees Celsius incubator for 24 hours. The next day, 10ul of media containing dilutions of compound in DMSO were added to each plate in triplicate with final concentrations of 0.025 uM PAV-617, 0.05 uM PAV-617, 0.1 uM PAV-617, 0.5 uM PAV-617, 0.02 uM PAV-951, 0.3 uM PAV-951, 0.4 uM PAV-951, or 0.5 uM PAV-951. Six wells on each plate received 10ul of media containing only DMSO. Each well was gently mixed 5 times with a 100ul pipette. Plates were incubated at 37 degrees Celsius for 72 hours then 10 uL of alamarBlue was added to each well. Wells were mixed 5 times then incubated at 37 degrees Celsius for 72 hours. Plates were then read at 530/590. Values were averaged and standard deviations were calculated for each triplicate condition and graphed on Microsoft Excel.

Cell growth recovery assay

A 96 well plate was seeded with either Hennes 20 or LNCaP C-33 cells at 500 cells/well in 90 uL of minimum essential media and incubated at 37 degrees Celsius for 24 hours. 0.5% DMSO was diluted in media and added to 6 control wells for each plate. PAV-617 was diluted in media and added to three wells at a concentration of 0.3 uM. PAV-951 was diluted in media and added to concentration of 0.4 uM. After 24 hours of PAV-617 treatment or 6 hours of PAV-951 treatment, the medium containing compound was removed and replaced with fresh media. After 72 hours (day 5), plates were assayed with alamarBlue and fluorescence was read at 530/590. The medium containing alamarBlue was removed and replaced with fresh media. After another 72 hours (day 8) plates were assayed with alamarBlue and fluorescence again, then medium containing alamarBlue was removed and replaced with fresh media. After a final 72 hour incubation (day 11) plates were assayed with alamarBlue one more time. Average fluorescence for each day and treatment condition was graphed on Prism and IC50s were calculated with the nonlinear regression [Inhibitor] vs. normalized response -- Variable slope.

Human tumor cell proliferation assay

A panel of human tumor cell lines (A172, BFTC-905, COR-L105, DB, FaDu, H9, Hs 294T, MCF7, MDA MB 436, MeWo, MHH-PREB-1, SJSA1-OSA, SW1353, and U2OS) were grown in RPMI 1640, 10% FBS, 2 mM L-alanyl-L-glutamine, 1 mM Na pyruvate. Cells were seeded into 384-well plates and incubated in a humidified atmosphere with 5% CO2 at 37°C. After 24 hours of incubation DMSO, PAV-617, or PAV-951 was added at concentrations of 5 uM, 1 uM, and 0.2 uM and plates were incubated for 3 days. Then cells were lysed with CellTiter-Glo (Promega) which generates a bioluminescence signal relative to ATP levels and is used as a measurement of viable cells. Bioluminescence was read by a PerkinElmer Envision microplate reader.  Bioluminescence intensity was measured by a PerkinElmer Envision microplate reader and transformed to a percent of control (POC) using the formula: POC=(Ix/I0)*100, where Ix is the whole well signal intensity at a given treatment, and I0 is the average intensity of the untreated vehicle wells. Data was graphed on Prism and IC50s were calculated with the nonlinear regression [Inhibitor] vs. normalized response -- Variable slope.

BB1 and BB5 Adenoma versus Carcinoma screen

Two cell lines, BB1 and BB5, were derived from two patient breast biopsies that were obtained prior to treatment. Follow up classification indicated that BB1 was a benign adenoma and BB5 was a breast adenocarcinoma. The cells were grown in conditional reprogramming media in co-culture with irradiated fibroblasts as described in Liu et al., 2017(69). Once cultures reached exponential growth phase, the cells were harvested and seeded. After 48 hours of growth, cells were treated with PAV-617, PAV-951, 0.1% DMSO (vehicle), or 1uM staurosporine (positive control which induced cell death) for 60 hours. Compound treated cells were imaged with Hoechst 33342, TMRE, and MultiChrome-3 which stained the nucleus, functional mitochondria, and cellular membranes respectively.

National Cancer Institute Sixty Cell Line Screen

In the NCI-60 screen, 59 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, PC-3, DU-145, MCF7, MDA-MB-231/ATCC, HS 578T, BT-549, T-47D, MDA-MB-468) 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).

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. Values are calculated for each of these three parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested.

Mouse maximum tolerated dose determination

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), 1mg/kg PAV-617, 2 mg/kg PAV-617, 5 mg/kg PAV-617, 10 mg/kg PAV-617, 1mg/kg PAV-951, 2.5 mg/kg PAV-951, 5mg/kg PAV-951 or 10 mg/kg PAV-951. 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.

For the oral MTD study, female CD1 mice aged 5-6 weeks were given either an oral dose of vehicle (10% DMSO, 45% propylene glycol, 45% sterile water) or either 10 mg/kg or 20 mg/kg of PAV-617 or PAV-951. The vehicle and 20 mg/kg groups had three animals each, while the 10 mg/kg groups only had one animal. Animals were observed for clinical signs and after a week they were euthanized and examined externally and internally by a pathologist for changes related to toxicity.

Pharmacokinetics studies

Male Sprague Dawley rats aged 8-10 weeks were randomly divided into treatment groups with four animals per group. Animals in each treatment group were weighed and received one 2.4 mL intravenous dose of either vehicle (10% DMSO, 45% propylene glycol, 45% sterile water), 1mg/kg PAV-617, or 0.5 mg/kg PAV-951, or one intraperitoneal dose of either vehicle (100% labrasol), 5mg/kg PAV-617 or 2.5 mg/kg PAV-951 . Blood was collected from a pre-cannulated line before dosing, and subsequently 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and 30 hours post-dosing. Concentration of drug in the plasma over time was measured using a Waters Acquity TQD LCMS/MS. Maximum concentration (Cmax) was determined to be the maximum concentration detected in a dataset. Half life, area under the curve, and mean residence time were calculated with Phoenix WinNolin software.

CD1 or Balb/c nude mice aged 6-8 weeks were randomly divided into treatment groups with three animals per group. Animals in each treatment group were weighed and received either one 2.4 mL intravenous dose of either vehicle (10% DMSO, 45% propylene glycol, 45% sterile water) or 2.5 mg/kg PAV-951, or one intraperitoneal dose of either vehicle (100% 10% DMSO, 45% propylene glycol, 45% sterile water) or 5mg/kg PAV-621. Animals were sacrificed after 1 hour, 4 hours, 8 hours, 24 hours post-dosing. Concentration of drug in the plasma and lung over time was measured using a Waters Acquity TQD LCMS/MS. Maximum concentration (Cmax) was determined to be the maximum concentration detected in a dataset. Half life, area under the curve, and mean residence time were calculated with Phoenix WinNolin software.

A549 xenograft studies

A549 cells growing in RPMI-1640 medium were suspended with Matrigel in PBS. 0.1 mL of cell suspension containing 1 x 10⁶ cells were injected subcutaneously into the left flank region of female, 6-8 weeks old nude mice (CrTac: Ncr-Foxn1nu). After 30 days of tumor establishment, mice were divided randomly into treatment groups. In the PAV-617 study, 6 animals were treated with vehicle only (10% DMSO, 10% propylene glycol, 80% sterile water) by IP once daily, 6 animals were treated with 100 mg/kg Gemcitabine Hydrochloride by IP twice weekly, and 6 animals were treated with 10 mg/kg PAV-617 by IP once daily for 28 days. In the PAV-951 study, 6 animals were treated with vehicle only (10% DMSO, 10% propylene glycol, 80% sterile water) by IV once daily, 6 animals were treated with 100 mg/kg Gemcitabine by IV twice weekly, and 6 animals were treated with 1.5 mg/kg PAV-951 by IV once daily for 14 days. In both studies, mice were weighed and their tumors were measured using a digital Vernier caliper. Tumor volume was calculated using the formula: (L x W2)/2 where L is the largest diameter and W is the smallest diameter of the tumor. Statistical analysis was performed using Graph Pad Prism (Ver. 5.03). Statistical analysis of tumor growth inhibition between the Control and Treated groups was performed by using One-way ANOVA followed by Dunnett’s test.

HT-29 xenograft study

HT-29 cells growing in HBSS medium were suspended in Matrigel. 0.1 mL of the cell suspension containing 5x10⁶ cells were injected subcutaneously into the left flank region of male, 6-8 week old SCID mice. After tumor establishment, animals were divided randomly into groups of 6 and treated daily by IV with vehicle daily for 17 days, 3 mg/kg PAV-951 three times per week for 17 days, or treated by IP with 60 mg/kg Irinotecan every 4 days for 14 days. Tumor volume was calculated using the formula: (L x W2)/2 where L is the largest diameter and W is the smallest diameter of the tumor. Statistical analysis was performed using Graph Pad Prism (Ver. 5.03). Statistical analysis of tumor growth inhibition between the Control and Treated groups was performed by using One-way ANOVA followed by Dunnett’s test.

Drug Resin affinity chromatography

A549 cells were grown in minimum essential media (UCSF) with 10% FBS and 1% Penstrep for 24 hours then treated with 500nM PAV-617, 500nM PAV-951, or DMSO for 22 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 4°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). In some conditions, 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 either 4oC or 22oC on 30 ul of affi-gel resin coupled to either PAV-617, methylene blue, PAV-951, PAV-621, or a 4% agarose matrix (control). The input material was collected and the resin was then washed with 3 mL column buffer. The resins were eluted overnight at either 4oC or at 22oC in 100ul column buffer containing either 100uM PAV-617, 100uM PAV-951, 100uM PAV-621 or 1% DMSO, with or without the energy cocktail. In some experiments, the depleted flow through from the resins were incubated on a second column, washed 100x in column buffer, and then stripped with 1% SDS. Eluates were run on SDS-PAGE with samples for silver stain and/or western blot or sent for mass spectrometry analysis.

Sucrose Step Gradients

Sucrose step gradients were performed as previously described, however due to the inclusion of metabolic energy in the eluates, the gradients were centrifuged at room temperature rather than 4 degrees Celsius (70).

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 KAP-1, MTHFD1, hnrnpK, TUBB, or PDI 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.

Tandem mass spectrometry

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 °C 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 and the data set was further adjusted by subtraction of spectral counts for specific proteins observed in the control resin. Identified proteins were searched in the Bushman labs oncogene database (http://www.bushmanlab.org/links/genelists) or the database as described in Jager et al., 2012 (30) to determine if they interact with HIV.