DOI: 10.5061/dryad.k3j9kd5hf
title: Data from: Small molecule protein assembly modulators with Pan-Cancer therapeutic efficacy

description: 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.
description: <strong>Chemical Synthesis</strong>
 All compounds synthesized were confirmed by LCMS with purity
 typically &gt; 98% <strong>Synthesis of
 PAV-617</strong> (see <strong>Supplemental Figure
 1A</strong>) <em>Phenothiazin-5-ium
 tetraiodide hydrate</em> 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%).
 <em>3,7-Di(pyrrolidin-1-yl) phenothiazinium
 iodide</em> 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. <strong>Synthesis of
 PAV-617 resin </strong>(see <strong>Supplemental Figure
 1B</strong>)
 <em>2-Aminomethylphenothiazine</em>
 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)
 <em>2-Bocaminomethylphenothiazine</em>
 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
 <em>2-Bocaminomethylphenothiazin-5-ium tetraiodide
 hydrate</em> 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.).
 <em>2-Bocaminomethyl-3,7-di(pyrrolidin-1-yl)
 phenothiazin-5-ium iodide</em> 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)
 <em>2-Aminomethyl-3,7-di(pyrrolidin-1-yl)
 phenothiazin-5-ium dihydrochloride</em>
 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.
 <strong>Synthesis of PAV-951 (see Supplemental Figure
 2A)</strong> 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). <strong>Synthesis of PAV-951
 resin</strong> (see <strong>Supplemental Figure
 2B</strong>) 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).
 <strong>Experimental Models and Subject
 Details</strong> <strong>Animal
 models</strong> 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 CO<sub>2</sub>. 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 5x10<sup>6</sup> 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. <strong>Cell
 lines</strong> 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. <strong>BB1 and BB5
 Adenoma versus Carcinoma screen</strong> 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.
 <strong>National Cancer Institute Sixty Cell Line
 Screen</strong> 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. <strong>In vitro
 experiments</strong> 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.
 <em>Method and Analysis Details</em>
 <strong>Mpox virus infection assay</strong>
 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. <strong>Human
 immunodeficiency virus infectious virus assay </strong>
 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.
 <strong>Apoptosis Screen</strong>
 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.
 <strong>High density/ low density
 assay</strong> 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.
 <strong>Cell growth recovery assay</strong>
 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. <strong>Human tumor
 cell proliferation assay</strong> 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. <strong>BB1 and BB5 Adenoma versus
 Carcinoma screen</strong> 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.
 <strong>National Cancer Institute Sixty Cell Line
 Screen</strong> 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 <em>in situ</em> 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 <em>in situ</em> 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&gt;/=Tz [(Ti-Tz)/Tz] x 100 for concentrations
 for which Ti&lt;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. <strong>Mouse
 maximum tolerated dose determination</strong> 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.
 <strong>Pharmacokinetics studies</strong>
 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. <strong>A549 xenograft
 studies</strong> A549 cells growing in RPMI-1640
 medium were suspended with Matrigel in PBS. 0.1 mL of cell suspension
 containing 1 x 10<sup>6</sup> 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.
 <strong>HT-29 xenograft study</strong>
 HT-29 cells growing in HBSS medium were suspended in Matrigel.
 0.1 mL of the cell suspension containing 5x10<sup>6</sup>
 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.
 <strong>Drug Resin affinity
 chromatography</strong> 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. <strong>Sucrose
 Step Gradients</strong> 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).
 <strong>Silver Stain</strong>
 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.
 <strong>Western blotting</strong>
 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. <strong>Tandem mass
 spectrometry</strong> 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.
description: # Small Molecule Protein Assembly Modulators with Pan-Cancer Therapeutic
 Efficacy
 [https://doi.org/10.5061/dryad.k3j9kd5hf](https://doi.org/10.5061/dryad.k3j9kd5hf) **File description:** Each tab in the Excel file contains the raw data corresponding to a specific figure. Tabs are labeled according to the figure number, with supplemental figures indicated by an 's' before the number.  In this Excel file, cells containing "n/a" represent missing or unavailable data for that particular entry. **Tab descriptions:** **Figure 1** 1A- shows assessment of the endogenous apoptosis response in multiple cell lines. Plates were seeded with LNCaP C-33, LNCaP C-81, CHO K-1, and Hennes 20 cells. After three days of growth Apo-ONE reagent was added and caspase-3/7 activity was determined by fluorescent readout. Excel document contains F521 absorbance (RFU) values for each cell line. 1D and 1E- show activity of PAV-617 and PAV-951 in the Hennes 20 “arrest of proliferation” assay where parallel plates of Hennes 20 cells were seeded at a high density of 15,000 cells per well and a low density of 500 cells per well and treated with DMSO, PAV-617, or PAV-951. Fluorescent reading (RFU) corresponding to cell viability was calculated using an AlamarBlueTM assay. The average absorbance of three wells containing only media was subtracted from the absorbance values of the vehicle and treatment groups. The resulting values were then divided by the average absorbance of six vehicle wells to calculate the percentage growth, shown in the excel document. 1F, 1G,1H, and 1I- show recovery of cancer cell growth following removal of compound. Hennes 20 or LNCaP C-33 cells were seeded at a low density and then incubated with DMSO, PAV-617, or PAV-951. After a period of treatment, the medium containing compound was removed and replaced with fresh media. Plates were assessed for cell viability by AlamarBlueTM on day 5, 8, 11, and 14. Excel document contains F590 absorbance (RFU) for vehicle and drug treated cells on each day. **Figure 2** 2A- shows activity of PAV-617 against MPXV. BSC-40 cells were infected with 100 plaque forming units of MPXV Zaire 79 and treated with PAV-617 for three days. In the excel document plaques observed are shown as a percentage of the plaques observed in untreated cells. 2B- shows activity of PAV-951 against HIV. MT-2 cells were infected with NL4-3 Rluc HIV and treated with PAV-951 for four days. In the excel document plaques observed are shown as a percentage of the plaques observed in untreated cells. **Figure 3** 3A- shows the percentage growth values of PAV-617 and PAV-951 assessed in house against A549, HT29, LNCaP C-33, and PANC-1 cell lines. These values were used to calculate the IC50’s shown in figure 3A of the paper. 3B- shows the average percentage growth values of PAV-617 and PAV-951 in the Eurofins OncoPanel against A172, BFTC-905, COR-L105, DB, FaDu, H9, Hs 294T, MCF7, MDA MB 436, MeWo, MHH-PREB-1, SJSA1-OSA, SW1353, U2OS, HMEC, and HUVEC cell lines. 3C- shows the mean optical density and percentage growth of PAV-617 and PAV-951 in the NCI-60 screen against 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 cell lines. These values were used by the NCI to calculate the IC50s and LC50s shown in figure 3C. 3D- shows the percentage growth values of PAV-617 and PAV-951 assessed on HMEC and HUVEC cell lines. **Figure 4** 4A- shows tumor volume (mm3) across time for an A549 lung cancer treated with vehicle, PAV-617, or Gemcitabine hydrocholoride. 4B- shows tumor volume (mm3) across time for a A549 lung cancer treated with vehicle, PAV-951, or Gemcitabine hydrochloride. 4C- shows tumor volume (mm3) across time for a HT-29 colorectal cancer treated with vehicle, PAV-951, or Irinotecan. **Figure 5** 5A, 5B, and 5C- shows optical density of various proteins western blots. **Figure 6** 6- growth percentages of PAV-541, PAV-621, PAV-951, and PAV-436 tested on the NCI-60 assay by the NCI. **Supplemental figures** s3- shows the percentage growth values of PAV-951, PAV-621, and gemcitabine against MRC5 and A549 cells. Fluorescent reading (RFU) corresponding to cell viability was calculated using an AlamarBlueTM assay. The average absorbance of three wells containing only media was subtracted from the absorbance values of the vehicle and treatment groups. The resulting values were then divided by the average absorbance of six vehicle wells to calculate the percentage growth, shown in the excel document. s7.1- shows safety data from an 11-day body weight study of mice treated with either a vehicle or 2.5 mg/kg PAV-951 s7.2- shows hematology of the treated mice upon completion of the study. s7.3- shows the concentration of PAV-951 or PAV-621 in the blood or lung of CD1 (PAV-951) or balb/c nude mice (PAV-621) following a single IV or IP injection. s7.4- show the concentration of PAV-617 and PAV-951 in rats following a single IV or IP dose of compound. s8A- shows spectral counts of proteins detected by MSMS in DRAC eluates from the PAV-617 resin under cold or hot conditions, as well as from the methylene blue resin and the negative control resin. s8B- shows spectral counts of proteins detected in the PAV-951 and PAV-621 resin eluates, as well as negative control resins eluted with PAV-951 and PAV-621. s9- shows optical density of ALDH1A1 western blot. s10- shows the percentage growth values of PAV-951, PAV-621, and gemcitabine against A549 cells at different treatment times. A549 cells were plated at a concentration of 2,500 cells/well and treated with 0.1uM, 0.5uM, 2.5uM or 12.5uM compound or vehicle concurrently with plating (“time 0”) or 24 hours later. After 72 hours, cell viability was measured by an AlamarBlueTM assay.