WO2021252099A2 - Antimicrobial and antitoxin compositions and methods for treatment - Google Patents

Abstract

Methods of treating coronavirus infections, ricin poisoning, and inhibiting growth of rod- shaped bacteria are provided. The methods include administering to a subject or contacting the bacteria with AR-12 or a derivative thereof or a multikinase inhibitor such as sorafenib tosylate or pazopanib, optionally in combination with one or more PDE5 inhibitors.

Description

ANTIMICROBIAL AND ANTITOXIN COMPOSITIONS AND METHODS FOR

TREATMENT

FIELD OF THE INVENTION

The invention is generally related to the use of AR-12 and derivatives thereof or multi kinase inhibitors, alone or in combination with PDE5 inhibitors, as anti-viral, anti-bacterial, and anti-toxin agents.

BACKGROUND OF THE INVENTION

The chaperone GRP78 / BiP / HSPA5 / Dna K is conserved throughout evolution down to prokaryotes ^[1]’ ^[2]. AR-12 (OSU-03012) was shown to reduce expression of the chaperone GRP78 through a process requiring autophagosome formation ^[3]. Subsequently, using quantitative immunofluorescent staining of single cells, it was determined that AR-12 rapidly caused epitopes at the NH2-termini of GRP78, HSP90 and HSP70 to become occluded, whereas epitopes at the COOH-termini were unaffected ^{|4| [5]}’ ^[6]’ ^[7]’ ^[8]’ ^[9]. AR-12 was then shown to inhibit the ATPase activities of GRP78, HSP90 and HSP70 with IC50 values in the clinically relevant 100-300 nM range ^[9]. In the clinic, AR-12 was safely dosed in heavily pre treated cancer patients at 800 mg BID (NCT00978523; ASCO 2013 meeting). The C max of AR-12 in plasma after 1 day at the MTD of 800 mg BID was ~2 mM. After 28 days of treatment the C max was ~3 mM with the peak C max in some patients being ~8 pM. Some patients were on this trial with stable disease for up to 9 months without any drug-related toxicities (DLTs).

AR-12 exposure rapidly decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1, receptors for Ebola / Marburg / Hepatitis A, Lassa fever, and Hepatitis B viruses, respectively ^{[4] [5] |6| [7]}’ ^[8]. Clinically achievable concentrations of AR-12 prevented the replication of drug-resistant HIV, Rabies, Junin, Coxsackievirus B4, Ebola, Chikungunya, Mumps, Measles, Rubella, RSV, CMV, and Influenza viruses. In three different animal models, AR-12 suppressed the production of infectious vims particles and prolonged animal survival ^{[6] [10] [11]}.

The coronavirus SARS-CoV-2 is the causal agent of the 2020 global COVID-19 pandemic. It is a novel non-human-derived vims whose biology in humans is completely unknown. The development of an anti-viral agent that could suppress SARS-CoV-2 reproduction is viewed as a high priority for biomedical researchers. Thus, new antiviral agents targeting coronaviruses and other pathogens are needed.

SUMMARY

Embodiments of the disclosure provide methods of treating coronavirus infections, ricin poisoning, and inhibiting growth of rod-shaped bacteria. The methods include administering to a subject in need thereof or contacting the bacteria with a compound of Formula I or a multikinase inhibitor, optionally in combination with one or more PDE5 inhibitors.

One aspect of the disclosure provides a method of treating a coronavirus infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising at least one compound or pharmaceutically acceptable salt, solvate or hydrate thereof having the general chemical structure of:

Formula I wherein

Xi is H, or an unbranched, branched, saturated, unsaturated, substituted, or unsubstituted C1-C8 alkyl group; and

X2 is an unbranched, branched, saturated, unsaturated, substituted, or unsubstituted C1-C8 alkylcarbonyl; substituted or unsubstituted aryl or heteroarylcarbonyl; substituted or unsubstituted alkylsulfonyl; carboxamide, sulfonamide, aminosulfonamide, acylamido, or H.

In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the method further comprises administering one or more PDE5 inhibitors. In some embodiments, the one or more PDE5 inhibitors is selected from the group consisting of sildenafil, vardenafil, tadalafil, avanafil, udenafil, microdenafil, and lodenafil carbonate. In some embodiments, the compound is selected from the group consisting of AR-12, AR-13, and AR-14. Another aspect of the disclosure provides a method of treating a coronavirus infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising sorafenib tosylate or pazopanib.

Another aspect of the disclosure provides a method of treating ricin poisioning in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a compound of Formula I or pharmaceutically acceptable salt, solvate or hydrate thereof. In some embodiments, the method further comprises administering one or more PDE5 inhibitors. In some embodiments, the one or more PDE5 inhibitors is selected from the group consisting of sildenafil, vardenafil, tadalafil, avanafil, udenafil, microdenafil, and lodenafil carbonate. In some embodiments, the compound is selected from the group consisting of AR-12, AR-13, and AR-14.

Another aspect of the disclosure provides a method of treating ricin poisioning in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising sorafenib tosylate or pazopanib.

Another aspect of the disclosure provides a method of inhibiting the growth of rod shaped bacteria, comprising contacting the rod-shaped bacteria with a compound of Formula I or pharmaceutically acceptable salt, solvate or hydrate thereof. In some embodiments, the rod-shaped bacteria is selected from the group consisting of Bacillus, Clostridium, Yersinia, and Klebsiella. In some embodiments, the method further comprises contacting the rod-shaped bacteria with one or more PDE5 inhibitors. In some embodiments, the one or more PDE5 inhibitors is selected from the group consisting of sildenafil, vardenafil, tadalafil, avanafil, udenafil, microdenafil, and lodenafil carbonate. In some embodiments, the compound is selected from the group consisting of AR-12, AR-13, and AR-14.

Another aspect of the dislosure provides a method of inhibiting the growth of rod shaped bacteria, comprising contacting the rod-shaped bacteria with sorafenib tosylate or pazopanib.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. AR-12 reduces SARS-CoV-2 spike protein expression in a dose-dependent fashion. HCT116 ATG16L1 T300 cells were transfected with a plasmid to express the SARS- CoV-2 spike protein. Twenty-four h later, cells were treated with vehicle control or with AR- 12 (100-2000 nM) for 6 h and fixed. Cells were stained to determine the expression of the SARS-CoV-2 spike protein and for ERK2 as a loading control (n = 3 ±SD).

Figures 2A-D. AR-12 suppresses the synthesis of the SARS-CoV-2 spike protein. A. Vero cells were treated with vehicle control or AR-12 and 6 h later infected with SARS-CoV- 2 as described in the Methods and Figure. Cells were fixed after 24 h or 48 h and the expression of the spike protein determined (n = 2 independent experiments each with 4 independent assessments +/-SD) *p < 0.05 less than corresponding values in vehicle control treated; ** p < 0.05 less than corresponding values in 1 mM AR-12 treated cells. B. Representative 60X images of drug-treated cells. C. and D. Vero cells were infected with SARS-CoV-2 and then treated with vehicle control or with AR-12 (2 mM), 3 h, 6 h and 12 h after infection. All cells were fixed 24 h after infection and the expression of the spike protein and GRP78 determined (n = 2 independent experiments each with 6 independent assessments ±SD) *p < 0.05 less than corresponding values in vims infected cells.

Figures 3A-C. AR-12 suppresses the expression of GRP78 and reduces the increase in GRP78 expression caused by SARS-CoV-2. A. Vero cells were pre-treated with vehicle control or with AR-12 for 6 h. Cells were infected with SARS-CoV-2 (10 MOI). Forty minutes after infection, the media was removed, and the cells washed with PBS three times. The cells were fixed in place without permeabilization and cells stained to determine the levels of spike protein on the outer leaflet of the plasma membrane, with DAPI and ERK2 as loading controls. B. Vero cells were pre-treated with vehicle control or with AR-12 for 6 h. Cells were then infected (10 MOI). The cells were fixed in place with permeabilization 10 min and 40 min after infection and the total expression of ACE2, GRP78, HSP90, HSP70, eIF2a, P-eIF2a S51 and ERK2 (not shown) determined (n = 2 independent experiments each with 4 independent assessments ±SD) *p < 0.05 less than corresponding values in vehicle control treated; ** p < 0.05 less than corresponding AR-12 value in uninfected cells; § p < 0.05 less than corresponding vehicle value in uninfected cells. C. Vero cells were treated with vehicle control or AR-12 (1 mM, 2 mM), sildenafil (2 pM) or the drugs combined and 6 h later infected with SARS-CoV-2 as described in the Methods and the Figure. Cells were fixed after 24 h or 48 h and the expression of GRP78 and ERK2 determined (n = 2 independent experiments each with 4 independent assessments ±SD) *p < 0.05 less than corresponding values in vehicle control treated; # p < 0.05 greater than corresponding value in uninfected cells. Figures 4A-B. Sorafenib and pazopanib modestly reduce (A) spike protein and (B) GRP78 expression in SARS-CoV-2 infected cells. Vero cells were infected with the indicated multiplicities of infection with SARS-CoV-2. One hour after infection, cells were treated with vehicle control, sorafenib tosylate (1.0 mM) or pazopanib (0.5 pM). Twenty-four h after infection, cells were fixed in place. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies ±SD).

Figures 5A-D. AR-12 inactivates eIF2a regardless of viral infection. A. Vero cells were treated with vehicle control or AR-12 (2 pM) and 6 h later infected with SARS-CoV-2 as described in the Methods. Cells were fixed after 24 h and the expression of eIF2a, phospho- eIF2a serine 51 and ERK2 determined (n = 2 independent experiments each with 4 independent assessments ±SD) # p < 0.05 greater than corresponding value in vehicle control treated cells. B. and C. HCT116 ATG16L1 T300 cells were transfected to express the SARS- CoV-2 spike protein and in parallel, transfected with a scrambled siRNA (siSCR) or an siRNA to knock down the expression of eIF2a. Twenty-four h later, cells were treated with vehicle control or AR-12 (2 pM) and 6 h later the cells fixed in place. The expression of spike protein, eIF2a and ERK2 were determined (n = 2 independent experiments each with 3 independent assessments ±SD) * p < 0.05 less than vehicle control value; # p < 0.05 greater than vehicle control value; § p < 0.05 greater than corresponding value in siSCR cells. D. HCT116 ATG16L1 T300 cells were transfected to express the SARS-CoV-2 spike protein and in parallel, transfected with a scrambled siRNA (siSCR) or an siRNA to knock down the expression of eIF2a. Twenty-four h later, cells were treated with vehicle control or AR-12 (2 pM) and 6 h later the cells fixed in place. The co-localization of viral spike protein and ATG5 was determined. The expression of Beclinl was presented pictorially.

Figures 6A-B. Representative images of GRP78 and SARS-CoV-2 spike protein expression and co-localization. A. and B. Vero cells were treated with vehicle control or AR- 12 (1 pM, 2 pM) and 6 h later infected with SARS-CoV-2 as described in the Methods. Cells were fixed after 24 h and the expression of GRP78 and ERK2 determined (n = 2 independent experiments each with 4 independent assessments +/-SD). Representative 60X images at the 24 h timepoint of the colocalization of the virus spike protein and GRP78. DAPI staining is the nucleus. Figures 7A-B. AR-12 suppresses the production of infectious SARS-CoV-2 virions. A. Four independent TCID50 / ml studies are presented, each study in independent quadruplicate. Vero cells were pre-treated with AR-12 and 6 h afterwards infected, and 24 h later the media was removed from the cells. The media was diluted 1:10, with repeated 1:10 dilutions using the Reed-Muench method. B. Four independent TCID50 / ml studies are presented, each study in independent quadruplicate. Vero cells were infected then after 6 h treated with AR-12, and 24 h later the media was removed from the cells. The media was diluted 1:10, with repeated 1:10 dilutions using the Reed-Muench method.

Figures 8A-E. AR-12 reduces the expression of SARS-CoV-2 spike protein, GRP78, HSP90 and HSP70 via autophagy. A. HCT116 cells (ATG16L1 A300 and T300) were transfected with a plasmid to express LC3-GFP-RFP. Twenty-four h after transfection, cells were treated with vehicle control or with AR-12 (2 mM). Cells were imaged 4 h and 8 h after drug exposure and the mean number of GFP + and RFP + punctae per cell determined from at least 40 cells per condition (n = 3 ±SD) # p < 0.05 greater than corresponding value in A300 cells. B. HCT116 ATG16L1 T300 cells were transfected with a plasmid to express the SARS- CoV-2 spike protein, and also transfected with a scrambled siRNA or siRNA molecules to knock down the expression of Beclinl or ATG5. Twenty-four h after transfection, cells were treated with vehicle control or AR-12 (2 pM). After a further 6 h incubation, cells were fixed and staining for the spike protein performed (n = 3 ±SD) * p < 0.05 less than corresponding value in siSCR cells. Images of the siRNA knockdown and AR-12 spike-suppressive effects are presented in Panel D at 60X magnification. C. HCT116 ATG16L1 T300 cells were transfected with a scrambled siRNA or siRNA molecules to knock down the expression of Beclinl or ATG5. Twenty-four h after transfection, cells were treated with vehicle control or AR-12 (2 pM). After a further 6 h incubation, cells were fixed and staining for GRP78, HSP90 and HSP70 performed (n = 3 ±SD) * p < 0.05 less than corresponding value in siSCR cells. E. HCT116 ATG16L1 T300 and A300 cells were transfected with a scrambled siRNA or siRNA molecules to knock down the expression of Beclinl or ATG5. Twenty-four h after transfection, cells were treated with vehicle control and after 6 h fixed in place. Upper: representative 60X images staining for the SARS-CoV-2 spike protein. Lower: representative 60X images staining for the co-localization of the SARS-CoV-2 spike protein and the endosomal protein LAMP2 in HCT116 ATG16L1 T300 cells. Figures 9A-D. HCT116 ATG16L1 T300 cells express greater amounts of GRP78 and ACE2 compared to cells expressing ATG16L1 A300 A. Vero, ADOR and HCT116 ATG16L1 T300 cells were plated and 24 h after plating, fixed and stained. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies ±SD). B. ADOR and Vero cells were treated with vehicle control or with AR-12 (2 mM) and 6 h later infected with SARS-CoV-2 (0.1 MOI, 0.01 MOI). Cells were fixed in place 24 h after infection. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies ±SD). * p < 0.05 less than corresponding value without virus infection; # p < 0.05 greater than corresponding value in ADOR cells. C. ADOR cells were treated with vehicle control or with AR-12 (1 mM, 2 pM). Cells were fixed in place after 6 h. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies ±SD). * p < 0.05 less than vehicle control. D. HCT116 ATG16F1 T300 and HCT116 ATG16F1 A300 cells were treated with vehicle control or with AR-12 (2 pM). Cells were fixed in place after 6 h. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression and phosphorylation of the indicated proteins (n = 3 independent studies ±SD). * p < 0.05 less than vehicle control; ¾ p < 0.05 less than corresponding value in T300 cells; # p < 0.05 greater than vehicle control; § p < 0.05 less than corresponding stimulated value in T300 cells.

Figures 10A-D. The weak infectivity of ADOR cells is associated with reduced TMPRSS2 and TMPRSS11D expression. A. and B. Vero, ADOR and HCT116 cells were infected with SARS-CoV-2 (1.0 MOI, 0.01 MOI) and the media collected 24 h, 48 h and 96 h after infection. The media was diluted 1:10, with repeated 1:10 dilutions using the Reed- Muench method. Vero cells were afterwards infected with the media, and 72 h later the media was removed from the cells and TCID50/ml values determined. C. ADOR cells were infected with SARS-CoV-2 and fixed 24 h after infection. Staining was performed to determine the localization of vims spike protein, GRP78 and nuclear DNA. A representative image is presented. D. Vero and ADOR were plated and 24 h after plating, fixed and stained. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies ±SD). * p < 0.05 less than corresponding values in Vero cells. Figure 11. AR-12 reduces the expression of PD-L1 and increases the expression of MHCA. HCT116 ATG16L1 T300 and A300 cells were treated with vehicle control or with AR-12 (2 mM). Cells were fixed in place after 6 h. Fixed cells were subjected to in-cell western blotting as described in the Methods to determine the expression of the indicated proteins (n = 3 independent studies ±SD). * p < 0.05 less than vehicle control: # p < 0.05 greater than vehicle control; ¾ p < 0.05 greater than corresponding value in T300 cells.

Figures 12A-C. Chemical Structures of (A) AR-12, (B) AR-13, and (C) AR-14.

Figures 13A-C. Sorafenib suppresses the growth of clinical isolate Klebsiella pneumoniae “blakpc superbug” bacteria and re-sensitizes bacteria to amplicillin. (A) Laboratory generic variant, 0.75 mM each. (B) and (C) Antibiotic resistant strains #1, #3, #4 (sorafenib, 2.0 pM each) in the presence or absence of 2.0 pg/ml ampicillin or 4.0 pg/ml meropenem and in the indicated combinations for 0-9h and 1 ml of media was removed and the bacterial isolated after centrifugation. The total amount of protein in the bacterial cell peppet was determined by Bradford assay (n = 3 +/-SEM).

DETAILED DESCRIPTION

Embodiments of the disclosure provide compositions containing AR-12 (OSU-03012) or derivatives thereof and/or multi-kinase inhibitors, optionally in combination with PDE5 inhibitors, that inhibit viral replication and bacterial growth and are a potent anti-toxin.

Embodiments of the disclosure provide compounds having the following general chemical structure of Formula I that are useful in the methods described herein.

Formula I wherein

Xi is H, or an unbranched, branched, saturated, unsaturated, substituted, or unsubstituted C1-C8 alkyl group; and X2 is an unbranched, branched, saturated, unsaturated, substituted, or unsubstituted Cl- C8 alkylcarbonyl; substituted or unsubstituted aryl or heteroarylcarbonyl; substituted or unsubstituted alkylsulfonyl; carboxamide, sulfonamide, aminosulfonamide, acylamido, or H.

As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that includes a fully saturated (no double or triple bonds) hydrocarbon group. The alkyl group may have 1 to 8 carbon atoms (whenever it appears herein, a numerical range such as "1 to 8" refers to each integer in the given range; e.g.,"l to 8 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 8 carbon atoms, although the present definition also covers the occurrence of the term "alkyl" where no numerical range is designated). The phrase “alkyl group” is intended to include not only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, tert-butyl, cycloalkyl and the like, but also alkyl substituents bearing further substituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl, halogen atoms, eyano, nitro, amino, carboxyl, etc. Thus, “alkyl group” includes ether groups, haloalkyls, nitroalkyls, carboxy alkyls hydroxyalkyls, cyanoalkyls, etc.

As used herein, “alkylcarbonyl” refers to carbonyl attached to the above alkyl.

As used herein, "alkoxy" refers to the formula -OR wherein R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl or a cycloalkynyl is defined as above. A non- limiting list of alkoxys is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso- butoxy, sec-butoxy and tert-butoxy. An alkoxy may be substituted or unsubstituted.

As used herein, "aryl" refers to a carbocyclic (all carbon) monocyclic or multicyclic aromatic ring system (including fused ring systems where two carbocyclic rings share a chemical bond) that has a fully delocalized pi-electron system throughout all the rings. The number of carbon atoms in an aryl group can vary. For example, the aryl group can be a Ce- Ci4 aryl group, a C6-C10 aryl group, or a Ce aryl group. Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene. An aryl group may be substituted or unsubstituted.

As used herein, “heteroaryl” is a heterocyclyl group derived from a heteroarene by removal of a hydrogen atom from any ring atom. Examples of heteroaryls include pyrrolidine, piperidine and pyridine.

“Carboxamide” refers to a moiety of the fomula -C(=0)NR^aR^b, where each of R^a and R^b is independently hydrogen, alkyl, cycloalkyl, aryl or aralkyl. As used herein, “sulfonyl” or “sulfuryl” refers to — S(0)₂ — .

As used herein, “sulfonamide” refers to — RSO2NH2 — a sulfone group connected to an amine group.

The term "acylamido" refers to CH3C(=0)NH.

It is understood that the methods and combinations described herein include crystalline forms (also known as polymorphs, which include the different crystal packing arrangements of the same elemental composition of a compound), amorphous phases, salts, solvates, and hydrates. In some embodiments, the compounds described herein exist in solvates forms with pharmaceutically acceptable solvents such as water, ethanol, or the like. In other embodiments, the compounds described herein exist in unsolvated form. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvents and may be formed during the process of crystallization with pharmaceutically acceptable solvent such as water, ethanol, or the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. In addition, the compound provided herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated form for the purpose of the compounds and methods provided herein.

In some embodiments, the compound is selected from AR-12, AR-13, or AR-14 (Figure 12A-C). OSU-03012 (AR-12; 2-Amino-A-[4-[5-phenanthren-2-yl-3-(trifluoromethyl)pyrazol- l-yl]phenyl]acetamide) is a derivative of celecoxib which no-longer acts against COX2 but instead inhibits the ATPase activity of multiple chaperone proteins, in particular GRP78. GRP78 acts as a sensor of endoplasmic reticulum stress and is an essential chaperone required for the life cycle of all mammalian viruses. AR-12 increases autophagosome formation and autophagic flux, enhances virus protein degradation, prevents virus reproduction, and prolongs the survival of infected animals.

Multikinase inhibitors work by inhibiting multiple intracellular and cell surface kinases, some of which are implicated in tumor growth and metastatic progression of cancer. The term "multiple kinase inhibitors" as used herein refers to any substance capable of simultaneously inhibiting the activity of at least two kinases, at least one or which is, for example, a tyrosine kinase. Non-limiting examples of kinases that may be inhibited include RAF, MEK (MAPK/ERK kinase), ERK (extracellular signal regulated kinase), ERBB2, and the like. Examples of multiple kinase inhibitors compatible with the present disclosure include, but are not limited to, sorafenib, pazopanib, regorafenib, lapatinib, trametinib, dabrafenib, vemurafenib, crizotinib, sunitinib, axitinib, ruxolitinib, vandetanib, cabozantinib, afatinib, ponatinib, ibrutinib, nintedanib, idelalisib, ceritinib, buparlisib, copanlisib, and the like.

The anti- viral or anti-bacterial or anti-toxin activity of compounds of Formula I or of the multikinase inhibitors as described herein may be significantly or synergistically enhanced when combined with one or more phosphodiesterase 5 (PDE5) inhibitors. PDE5 inhibitors were originally developed as agents to manipulate cardiovascular biology that were in parallel noted to treat erectile dysfunction. Suitable PDE-5 inhibitors include, but are not limited to, sildenafil, vardenafil, tadalafil, avanafil, udenafil, microdenafil, lodenafil carbonate, active components of Epimedium extracts, and other natural and synthetic analogues as described e.g. in Patel et ak, Screening of synthetic PDE-5 inhibitors and their analogues as adulterants: Analytical techniques and challenges, J. Pharm. Biomed. Anal. 87 (2014) 176-190, herein incorporated by reference; and in US patent application 20150231092, the complete contents of which is hereby incorporated by reference.

The compositions described herein (i.e. compounds of Formula I and/or multi-kinase inhibitors, optionally in combination with PDE5 inhibitors) are useful in methods of treating coronavirus infections. The term “treat/treating” refers to eliciting the desired biological response, i.e., a therapeutic effect which comprises one or more of a decrease/reduction in infection or infection symptom, a decrease/reduction in the severity of the infection (e.g., reduction or inhibition of viral adsorption, reduction or inhibition of viral replication, etc.), and an increased survival time of the affected host animal, following administration of the agent/composition.

Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans and birds, they cause respiratory tract infections that can range from mild to lethal. Mild illnesses in humans include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses), while more lethal varieties can cause SARS, MERS, and COVID-19. Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae , order Nidovirales, and realm Riboviria. They are enveloped viruses with a positive-sense single- stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses ranges from approximately 26 to 32 kilobases. The genome is wrapped with the nucleocapsid protein (N) inside an envelope consisting of three other structural proteins, i.e. the membrane protein (M), the envelope protein (E), and the spike protein (S). The S protein mediates the virus entry into the host cell via binding to the host angiotensin-converting enzyme 2 (ACE2) receptors. Exemplary coronaviruses that may be treated with the compositions of the disclosure include, but are not limited to, SARS-Cov, SARS-Cov-2, MERS-Cov, HCoV-OC43, HCoV-HKUl, HCoV-229E, and HCoV-NL63.

The compositions described herein (i.e. compounds of Formula I and/or multi-kinase inhibitors, optionally in combination with PDE5 inhibitors) are useful in methods of inhibiting the growth of bacteria, killing bacteria, or treating bacterial infections. In particular, the agents described herein have anti-bacterial activity against rod-shaped bacteria. Rod-shaped bacteria that may be targeted with the methods disclosed herein include, but are not limited to, Bacillus (e.g. B. anthraci ), Clostridium (e.g. C. botulinum), Yersinia (e.g. Y. pestis), and Klebsiella (e.g. K. pneumoniae).

Additional embodiments provide a method of sensitizing antibiotic resistant bacteria to an antibiotic, comprising contacting the antibiotic resistant bacteria with a composition as described herein, wherein said step of contacting renders said antibiotic resistant bacteria sensitive to said antibiotic.

The compositions described herein (i.e. compounds of Formula I and/or multi-kinase inhibitors, optionally in combination with PDE5 inhibitors) are useful in methods of treating ricin poisoning. Ricin is a highly toxic protein extracted from the seeds of the castor oil plant, Ricinus communis, belonging to the type II ribosome-inactivating protein family. Ricin has N-glycosidase activity and inhibits protein synthesis in mammalian cells, resulting in cell apoptosis and death. Therefore, ricin has been used as a biological warfare agent since the early 20th century, due to its high toxicity, easy extraction and high stability. Ingestion of ricin causes pain, inflammation, and hemorrhage in the mucosal membranes of the gastrointestinal system. Gastrointestinal symptoms quickly progress to severe nausea, vomiting, diarrhea, and difficulty swallowing (dysphagia). Haemorrhage causes bloody feces (melena) and vomiting blood (hematemesis). The low blood volume (hypovolemia) caused by gastrointestinal fluid loss can lead to organ failure in the pancreas, kidney, liver, and GI tract and progress to shock. Shock and organ failure are indicated by disorientation, stupor, weakness, drowsiness, excessive thirst (polydipsia), low urine production (oliguria), and bloody urine (hematuria). Symptoms of ricin inhalation are different from those caused by ingestion. Early symptoms include a cough and fever.

The methods of the disclosure involve administering compositions comprising at least one (i.e. one or more) of the drugs/compounds disclosed herein to a patient in need thereof. The drugs may be administered simultaneously or sequentially; separately or together in the same formulation. The present disclosure thus also provides compositions which comprise the compounds as described herein, usually together with a pharmacologically suitable carrier or diluent. In some embodiments, one substantially purified compound is present in a composition; in other embodiments more than one compound is present, each compound being substantially purified prior to being mixed in the composition.

Without being so limited, the medicaments/pharmaceutical compositions of the disclosure may be administered orally, for example in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. Administration can also be carried out rectally, for example using suppositories; locally, topically, or percutaneously, for example using ointments, creams, gels or solutions; or parenterally, e.g., intravenously, intramuscularly, subcutaneously, intrathecally or transdermally, using for example injectable solutions. Furthermore, administration can be carried out sublingually, nasally, or as ophthalmological preparations or an aerosol, for example in the form of a spray, such as a nasal spray.

The active agent may be combined with pharmaceutically acceptable excipients/carriers. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The medicaments/pharmaceutical compositions may also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents or antioxidants.

As used herein, "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention. These: salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid-addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-.beta.-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p- toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, 1-19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N-methyl- glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like.

Precursors (generally inactive precursors) of the compounds which are metabolized after administration to yield the compounds/active agents described herein in an active form are also encompassed.

A patient or subject to be treated by any of the compositions or methods of the present disclosure can mean either a human or a non-human animal including, but not limited to dogs, horses, cats, rabbits, gerbils, hamsters, rodents, birds, aquatic mammals, cattle, pigs, camelids, and other zoological animals. The compositions can also be administered in combination therapy, e.g., combined with at least one other agent able to treat or inhibit viral or bacterial infections or ricin poisoining.

In some embodiments, the active agent is administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of active agent to treat the infection, disease, or disorder at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the infection or disorder being treated and the severity of the infection disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific active agent employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels or frequencies lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage or frequency until the desired effect is achieved. However, the daily dosage of the active agent may be varied over a wide range from 0.01 to 1,000 mg per adult per day. In particular, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, in particular from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

In some embodiments, the compounds described herein are used prophylactically, e.g. they are administered to persons who have not yet exhibited symptoms of the disease or infection but are deemed to be at risk for developing the disease or infection (e.g. those who have been exposed to someone who is infected), or simply those who are at risk due to other factors, or those who may have been exposed to an infectious microbial agent or ricin. The compounds may also be administered to individuals who are thought or deemed to be exhibiting early signs of infection or disease or to be in early stages of infection or disease. The compounds may also be administered to individuals who are known to have and who definitely exhibit symptoms of infection or disease. Administration of the compounds described herein may prevent disease or infection symptoms, may slow the progression of disease or infection, and/or may reverse symptoms.

Before exemplary embodiments of the present invention are described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. -li lt is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely,"

"only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention.

EXAMPLE 1

Summary

In a dose-dependent fashion AR-12 inhibited SARS-CoV-2 spike protein expression in transfected or infected cells. AR-12 suppressed the production of infectious virions via autophagosome formation, which was also associated with degradation of GRP78. After AR- 12 exposure, the colocalization of GRP78 with spike protein was reduced. Knock down of eIF2a prevented AR-12-induced spike degradation and knock down of Beclinl or ATG5 caused the spike protein to localize in LAMP2+ vesicles without apparent degradation. HCT116 cells expressing ATG16L1 T300, found in the majority of persons of non-European descent, particularly from Africa, expressed greater amounts of GRP78 and SARS-CoV-2 receptor angiotensin converting enzyme 2 compared to ATG16L1 A300, predominantly found in Europeans, suggestive that ATG16L1 T300 expression may be associated with a greater ability to be infected and to reproduce SARS-CoV-2. In conclusion, our findings demonstrate that AR-12 represents a clinically relevant anti- viral drug for the treatment of SARS-CoV-2. Materials and Methods Materials In Mobile, the USA-WA1 strain of SARS-CoV-2 virus was acquired from BEI resources (Manassas, VA) and used to establish master and working stocks on Vero E6 (African green monkey kidney) cells. Vero E6 cells were acquired from the ATCC (Manassas, VA) and cultured in DMEM (Lonza), supplemented with 10% FBS (Millipore), lx L- glutamine (Lonza), and lx penicillin-streptomycin (Lonza). Vims stocks were verified to be free of mycoplasma contamination and infectious virus titers were determined by standard plaque assay on Vero E6 cells. AR-12 (OSU-03012), sorafenib tosylate and pazopanib were purchased from Selleck Chem (Houston, TX). In Richmond, Trypsin-EDTA, DMEM, RPMI, penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). Antibody to detect the SARS-CoV-2 spike protein and the plasmid to express the spike protein were from Sino Biological (Wayne, PA). Antibodies to detect Beclinl, ATG5, HSP90, HSP70, ERK2 and GRP78 were from Cell Signaling (Danvers, MA). ACE2, ODC, TMPRSS2-FITC and TMPRSS11D-AF647 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). Total ERK2, HSP90, HSP70, GRP78, eIF2 a, P-eIF2 a S51, ATG5, Beclinl, LAMP2, ATG13, P-ATG13 S318 and IDOl antibodies were from Cell Signaling (Danvers, MA). Anti-PD-Ll, PD-L2 and MHCA antibodies were from ABCAM (Cambridge, UK). Molecules to knock down the expression of Beclinl, ATG5 and eIF2 a, and scramble control, were purchased from Qiagen (Hilden, Germany). Female ADOR cells were a kind gift from a non-small cell lung cancer patient who, without any chemotherapeutic intervention, remains disease free five years after surgical removal of the tumor. The cells contain no ‘hot-spot” mutations. HCT116 ATG16L1 T300 and HCT116 ATG16L1 A300 cells were a kind gift from Dr. David L. Boone, Department of Microbiology and Immunology, Indiana University School of Medicine-South Bend, South Bend, IN ^[12]. Determination of antiviral activity and TCID50 values

TCID50 values were calculated by the Reed-Muench method ^[13]. To assess antiviral activity of AR-12, Vero E6 cells were seeded into 96 well tissue culture plates at 1.7 x 10⁴ cells/well and incubated overnight at 37 °C with 5% CO2. When cells reached >80% confluence (18 to 24 h) the cells were treated with AR-12 at the targeted concentration (1 or 2 mM) in viral growth media [DMEM supplemented with 2% FBS, lx L-glutamine and 0.2% DMSO (Fisher)]. Control wells received viral growth media alone. After 6 h, the cells were challenged with SARS-CoV-2 vims e.g. at a MOI of 0.001 (1.7 x 10¹ PFU/well) or 0.01 (1.7 x 10² PFU/well) and returned to the incubator at 37 °C with 5% CO2. Concentration of drug and DMSO remained constant in control and treated wells during challenge and each condition was tested in 4 wells/plate. At 24- or 48-hours post-infection, culture supernatant from each well was transferred to tubes for storage at -80 °C and cells were fixed by flooding the plates with 10% neutral buffered formalin. After 1 h, the formalin was removed and replaced with sterile PBS prior to immunofluorescence analysis. For therapeutic assessment, assay conditions remained the same except that the cells were infected first then treated with drug at 3-, 6- or 12-hours post-infection.

For TCID50 analysis, culture supernatant from each individual assay well was aliquoted to 3 wells of a 96 well plate containing DMEM supplemented with 2% FBS, lx penicillin-streptomycin, and lx L-glutamine. Ten-fold serial dilutions were performed for each sample and 100 pi of each dilution was transferred to the corresponding well of a 96 well tissue culture plate containing Vero E6 cells. After approximately 72 h incubation at 37 °C with 5% CO2, the wells from each dilution series were scored for the presence of cytopathic effects and the TC ID so/ml titer was calculated using the Reed-Muench method.

Assessments of protein expression and protein phosphorylation

In-cell western blotting was performed using the Hermes WiScan wide-field microscope ^{[14] [15]}. HCT116 cells (ATG16L1 T300 and A300) are sub-cultured into individual 96-well plates. Twenty-four hours after plating, the cells are transfected with a control plasmid or a control siRNA, or with an empty vector plasmid or with a plasmid to express the SARS- CoV-2 spike protein. After another 24 h, the cells are treated with vehicle control or AR-12. At various time-points after the initiation of drug exposure, cells are fixed in place using paraformaldehyde and using Triton X100 for permeabilization. Standard immuno-fluorescent blocking procedures are employed, followed by incubation of different wells with a variety of validated primary antibodies and subsequently validated fluorescent-tagged secondary antibodies are added to each well. Of note for scientific rigor is that the operator does not personally manipulate the microscope to examine specific cells; the entire fluorescent accrual method is independent of the operator. The Hermes microscope software randomly assesses the fluorescence intensity of 100 cells per treatment condition generating a mean fluorescence intensity read-out value to the investigator. The experiment is performed two additional times to provide an n = 3 (300 cells randomly analyzed). Transfection of cells

Cells from a fresh culture growing in log phase were transfected 24 h after plating. Prior to transfection, the medium was aspirated, and serum-free medium was added to each plate. For transfection, 10 nM of the annealed siRNA, scrambled or against eIF2 a, Beclinl or ATG5 were used. Ten nM siRNA (scrambled or experimental) was diluted in serum-free media. Four pi Hiperfect was added to this mixture and the solution was mixed by pipetting up and down several times. This solution was incubated at room temp for 10 min, then added drop-wise to each dish. The medium in each dish was swirled gently to mix, then incubated at 37 °C for 2 h. Serum-containing medium was added to each plate, and cells were incubated at 37 °C for 24 h before drug treatment.

Assessments of autophagosome and autolysosome levels

Cells were transfected with a plasmid to express LC3-GFP-RFP (Addgene, Watertown MA). Twenty-four h after transfection, cells are treated with vehicle control or AR-12 (2 mM). Cells were imaged at 60X magnification 4 h and 8 h after drug exposure and the mean number of GFP+ and RFP+ punctae per cell determined from >50 randomly selected cells per condition.

Quantification of phagocytosis

Phagocytosis was calculated at 15- and 60 min post-addition using flow cytometry analysis as described in Martinez et al. ^[16]. The percentage of phagocytosis equals the number of macrophages that have been engulfed PKH26-stained apoptotic cells, OVA-Alexa Fluor 594 (Invitrogen #034783), or Zymosan-Alexa Fluor 594 (Invitrogen #Z23374). Data represent a minimum of 2 independent experiments in which technical triplicates of 50,000 cells per sample were acquired using a Fortessa cytometer (BD).

Data analysis

Comparison of the effects of various treatments was done using one-way ANOVA and a two tailed Student’s z-test. Differences with a p-value of < 0.05 were considered statistically significant. Experiments are the means of multiple individual discrete points from multiple experiments (±SD).

Results

We initially performed a dose-response curve with AR-12, examining its ability to down-regulate the expression of the SARS-CoV-2 spike protein. Cells were transfected to express the spike protein and then treated with increasing concentrations of AR-12. In a dose- dependent fashion AR-12 reduced spike protein expression without altering the expression of the loading control ERK2 (Fig. 1).

Vero cells were treated with AR-12 (1 mM; 2 mM) and then infected with SARS-CoV- 2 at 0.01 and 0.001 multiplicities of infection (MOI). Twenty-four and forty-eight hours after infection, cells were fixed in place and permeabilized, and stained for expression of the S ARS- CoV-2 spike protein, total GRP78 and total ERK2 as a loading control. In a dose-dependent fashion, AR-12 suppressed the production of virus spike protein (Fig. 2 A and B). Cells were then infected and treated with AR-12 (2 pM) 3 h, 6 h and 12 h after infection, with cells being fixed and stained 24 h after infection. Treatment of infected cells with AR-12 significantly reduced the amount of spike protein produced in the infected cells as well as the amount of GRP78 in the cells (Fig. 2C and D).

Vero cells were treated with AR-12 (2 pM) and 6 h later infected with SARS-CoV-2 at 10 MOI. Forty minutes after infection, the cells were washed and then fixed in place, and the amount of virus spike protein associated with the Vero cell plasma membrane determined (Fig. 3 A). Prior AR-12 exposure reduced the amount of vims associated with the Vero cell plasma membrane by >75% (p < 0.05). Prior treatment for 6 h and 10 min with AR-12 significantly reduced the total expression of GRP78 (Fig. 3B). AR-12 did not or very weakly reduced the expression of ACE2 at the (6 h + 10 min) and (6 h + 40 min) time points, respectively. AR-12 did not or very weakly reduced the expression of HSP90 and HSP70 at the (6 h + 10 min) and (6 h + 40 min) time points, respectively. This data implies that AR-12, acting both as a direct inhibitor of GRP78 and by causing GRP78 break down, promotes the denaturation and inactivation of the ACE2 receptor; the denatured receptor cannot bind to the SARS-CoV-2 virus. Infection of cells with 0.01 MOI of SARS-CoV-2 caused the total expression of GRP78 to be significantly enhanced, 24 h/48 h after infection, whilst the expression of ERK2 remained constant (Fig. 3C). AR-12, in a dose-dependent fashion, significantly reduced basal GRP78 levels and almost abolished virus-stimulated GRP78 expression. Previously, we had also shown the multi-kinase and chaperone inhibitors sorafenib and pazopanib could suppress vims reproduction ^[7]. However, compared to AR-12, neither drug as strongly reduced spike protein nor GRP78 expression (Fig. 4).

GRP78 binds to and keeps inactive PKR-like endoplasmic reticulum kinase (PERK). PERK phosphorylates and inactivates eIF2a; for a vims, increased eIF2a phosphorylation will reduce translation of virus proteins, and our findings that vims infection increases GRP78 expression is concordant with this concept. AR-12 increased the inhibitory serine 51 phosphorylation of eIF2a regardless of viral infection (Fig. 5 A). Interestingly, virus infection increased the total expression of eIF2a, which was enhanced by AR-12. Knock down of eIF2a prevented AR-12 from reducing spike protein expression (Fig. 5B and C). Notably, when expression of eIF2a was knocked down, the spike protein became localized in punctate structures that co-stained for ATG5 (Fig. 5C and D). Although AR-12 increased expression of Beclinl, in the absence of eIF2a, AR-12 surprisingly reduced Beclinl levels (Fig. 5C and D). Similar data were also observed for ATG5. The chaperone GRP78 colocalized with the virus spike protein, an interaction disrupted by AR-12 (Fig. 6 A and B). Under control conditions the spike protein and GRP78 colocalize, and the merged image is yellow. In the presence of AR-12, not only is there less spike protein and GRP78 protein, but the colocalization color is now a green shade of yellow, indicating a greater degree of degradation of GRP78 than of the spike protein.

We then determined whether the AR- 12-dependent effects we had observed on the expression of vims spike protein and on GRP78 levels were associated with reduced production of infectious virus. The supernatants of infected cells, pre-treated with AR-12 for 6 h, were isolated 24 h after infection, and the 50% tissue culture infectious dose (TCID50) / ml titers determined. In a dose-dependent fashion, AR-12 significantly reduced the production of infectious SARS-CoV-2 (Fig. 7 A). The data presented are from four independent studies and within each study containing four independent determinations. In parallel, supernatants of infected cells, treated with AR-126 h after infection, were isolated 24 h after infection, and the TCID5o/ml titers determined. AR-12 significantly reduced the production of infectious SARS-CoV-2 (Fig. 7B). The data presented are also from four independent studies and within each study containing four independent determinations·

During the COVID-19 pandemic, African-Americans to a greater extent than European- Americans have been infected and killed by SARS-CoV-2. Many environmental and societal factors have been suggested to play causal roles in this epidemiology. One factor we have considered is the differential isoform expression of the autophagy regulatory protein ATG16L1 between African-Americans and European-Americans ^{| l 7| [18]}’ ^[19]’ ^[20]’ ^[21]’ ^[22]. African-Americans have been found to express ATG16L1 T300 to a greater extent than European-Americans who more frequently express ATG16L1 A300; homozygous expression of ATG16L1 A300 is associated with a greater incidence of Crohn’s Disease, due to a reduced ability of immune cells to phagocytose and digest, via autophagy, inflammatory materials in the GI. On the other hand, expression of ATG16L1 T300 has been linked to the greater metastatic spread of tumors ^[19]. The ability of AR-12 to stimulate autophagosome formation and cause autophagic flux was reduced in cells expressing the ATG16L1 A300 isoform (Fig. 8A) ^{[4] [6] [7]}. The ability of AR-12 to reduce expression of the SARS-CoV-2 spike protein required autophagosome formation (Fig. 8B). In cells expressing ATG16L1 T300, AR-12 significantly reduced spike protein expression by 71% whereas in cells expressing ATG16L1 A300, AR-12 only reduced spike protein levels by 16% (p < 0.05). The ability of AR-12 to reduce the expression of GRP78, HSP90 and HSP70 also required autophagosome formation (Fig. 8C and D).

In vehicle control treated cells, we noted that knock down of either Beclinl or ATG5, regardless of ATG16L1 isoform expression, altered the sub-cellular localization of the spike protein, from a diffuse mackerel stain to a more punctate form of staining (Fig. 8E, upper). Exploratory studies then determined whether knock down of the autophagosome regulatory proteins caused the spike protein to become localized in endosomes/lysosomes. After knock down of either ATG5 or Beclinl in HCT116 ATG16L1 T300 cells, the spike protein under basal conditions co-localized with the endosome marker LAMP2 (Fig. 8E, lower). Treatment of scrambled control cells with AR-12 reduced spike expression and caused punctate co localization of LAMP2 and Beclinl. Knock down of Beclinl or ATG5 both increased basal levels of LAMP2 by -25% (p < 0.05).

We compared the plasma membrane / surface (S) levels of the SARS-CoV-2 ACE2 receptor in Vero cells, a PDX non-small cell lung cancer isolate ADOR and HCT116 ATG16L1 T300 cells. The ADOR cells expressed approximately twice the amount of ACE2 compared to Vero cells and the HCT116 cells expressed approximately five times the amount of (Fig. 9A). We then compared the ability of ADOR cells and Vero cells to be infected and produce spike protein in the presence or absence of AR-12. Both cells expressed similar amounts of GRP78 and ERK2, and in both cells AR-12 reduced GRP78 expression (Fig. 9B). However, ADOR cells, despite being infected with ten-times more virus particles than the Vero cells, modestly expressed the SARS-CoV-2 spike protein. AR-12 significantly reduced the total and cell surface levels of GRP78 and ACE2 in ADOR cells whereas AR-12 did not reduce the expression of the ACE2 vims receptor in Vero cells (Fig. 9C). Comparing isogenic matched HCT116 ATG16L1 T300 and HCT116 ATG16L1 A300 cells, we found the expression of cell surface (S) and total expression of GRP78 (T) was reduced by approximately 25% in the HCT116 ATG16L1 A300 cells (Fig. 9D). The expression levels of ACE2 were also approximately 20% lower in the HCT116 ATG16L1 A300 cells. AR-12 reduced the expression of GRP78 and ACE2 and increased the phosphorylation of ATG13 S318 and eIF2a S51. However, the ability of AR-12 to increase the phosphorylation of ATG13 and eIF2a was significantly lower in the HCT116 ATG16L1 A300 cells. This is in further agreement with the reduced ability of the A300 cells from forming autophagosomes and performing autophagic flux.

Infection of HCT116 cells with SARS-CoV-2 did not induce any obvious cytopathic effect in either the T300 or the A300 cells within 96 h, and as assessed by TCID50 / ml assays. ADOR cells were capable of producing modest amounts of infectious virions (Fig. 10A and B). Thus, the levels of ACE2 in a cell do not correlate to the infectivity and viral reproduction capacity of cells. In a similar manner to our prior analyses, we determined whether the SARS- CoV-2 spike protein and the chaperone GRP78 co-localized in ADOR cells (Fig. IOC). Twenty-four hours after infection, some of the cells exhibited GRP78 and spike protein co localization, as judged by the cells staining orange. Portions of the cell population remained red, i.e. expressing GRP78 without spike protein. However, in cells expressing the highest levels of spike protein, low levels of GRP78 were present. DAPI staining for cells expressing high levels of spike protein revealed that the nuclear DNA was no-longer contained within an ovoid body, i.e. the cells had been killed by vims infection. Thus, ADOR cells contain at least two distinct populations of cells who respond differentially to SARS-CoV-2.

Our spike protein expression data comparing Vero cells and ADOR cells raised the possibility that the ability of SARS-CoV-2 to enter ADOR cells and HCT116 cells was compromised compared to that found in Vero cells. Trans-membrane serine proteases: serine subfamily member 2 (TMPRSS2) and TMPRSS11D, have been shown to play a key role in cleaving the SARS-CoV S protein at residues R667 and R797 ^[23]. These proteases played an essential role in the life cycle of the coronavirus SARS-CoV. Subsequently, similar data were obtained for SARS-CoV-2 ^{[24] [25]}. Compared to Vero cells, ADOR cells and HCT116 cells expressed significantly lower levels of both TMPRSS2 and TMPRSS11D (Fig. 10D). This data implies that the expression levels of S protein serine proteases may represent a better biomarker for SARS-CoV-2 infection and reproduction than the expression of the vims receptor itself, ACE2.

Checkpoint inhibitory immunotherapy antibodies, particularly those that inhibit PD- Ll, are a standard of care approach in diseases such as melanoma and non-small cell lung cancer. Treatment of HCT116 ATG16L1 T300 or A300 cells with AR-12 modestly reduced the expression of PD-L1 (Fig. 11 ). In T300, but not A300, cells, AR-12 treatment increased expression of class I MHCA, however, ATG16L1 A300 cells expressed higher basal levels of MHCA. Prior studies in solid tumor cells, and our present work in ADOR and HCT116 cells demonstrated that AR-12 could reduce the level of plasma membrane virus receptors. We determined whether AR-12, in immune cells, could enhance the phagocytosis of extracellular antigens. AR-12 did not significantly enhance antigen uptake in immune cells, regardless of Rubicon expression.

Discussion

Our data demonstrates that AR-12 suppresses the ability of SARS-CoV-2 to produce virus spike protein and to generate infectious virions. AR-12 reduces the expression of cell surface ACE2 and GRP78, as well as total GRP78 levels. We have previously demonstrated that AR-12-induced GRP78 degradation is causal in the formation of autophagosomes. Thus, AR-12 both catalytically inhibits the GRP78 ATPase activity, reducing its ability to renature proteins in the endoplasmic reticulum, and decreases the protein levels of the chaperone, all of which are associated with lower amounts of spike protein and infectious vims production. These data support the re-entry of AR-12 into the clinic as an anti-SARS-CoV-2 therapeutic.

One of the unexpected findings in our studies was the observation that in the absence of Beclinl or ATG5, the SARS-CoV-2 spike protein localized in endosomes/lysosomes. Of note, however, was that the protein levels of the spike protein had not declined. Hence, although the spike localized in a LAMP2 + compartment, those vesicles had not become acidified, i.e. no protein degradation had taken place. These findings suggest that autophagic flux is required to trigger LAMP2+ vesicle acidification, and resultant spike protein degradation. The malaria drug hydroxy chloroquine has been put forward as a therapeutic which can be used to treat COVID-19 patients. Hydroxy-chloroquine acts by preventing autophagic flux, leading to a build-up of autophagosomes. Hence, theoretically, if hydroxy chloroquine also causes translocation of the SARS-CoV-2 to LAMP2+ vesicles, a potential use for the dmg against this virus may be found i.e. a high initial dose of hydroxy-chloroquine drives spike protein into LAMP2+ vesicles concomitant with abolition of autophagic flux. Following removal and wash out of the drug, autophagic flux begins again, LAMP2+ vesicles containing spike protein acidify, and the spike protein is degraded. Of note, however, are findings that hydroxy-chloroquine use is not beneficial in severely infected patients ^[26].

In prior studies in the oncology field, we have shown that eIF2a phosphorylation is causal in the increased expression of Beclinl and ATG5 ^{[5] |6| [15]}. Those proteins were, in turn, essential for the formation of autophagosomes. Knock down of eIF2a, Beclinl or ATG5 prevented AR-12 from reducing spike protein expression. Thus, catalytic inhibition of GRP78 by AR-12 leads to increased eIF2a phosphorylation which enhances Beclinl and ATG5 expression, which promotes autophagosome formation leading to the degradation of GRP78 and spike protein.

There are relatively few biomarkers that can be used to define altered biology between different populations of humans. One such biomarker is ATG16L1 ^{[17] [18] [19] [20]}’ ^{[21] [22]}. In our oncology studies, expression of the isoform most commonly found in African Americans, ATG16L1 T300, predicted for enhanced tumor cell killing when treating the cells with agents that utilize autophagosome formation as a component of their killing mechanism ^[14]. However, drug combination treatments such as gemcitabine and paclitaxel, as used to treat pancreatic cancer, utilize autophagy as a survival mechanism; African Americans respond less effectively to this drug combination than European Americans ^[27]’ ^[28]. We discovered that cells homozygous for ATG16L1 T300 expressed approximately 20% more of the SARS-CoV- 2 receptor ACE2, as well as approximately 25% higher levels of GRP78. These findings would predict for greater infectivity comparing cells expressing the T300 isoform to cells expressing the A300 isoform. However, cells expressing the A300 isoform were less capable of causing ATG13 and eIF2a phosphorylation. Clinical samples from colorectal cancer patients homozygous for ATG16L1 A300 had elevated expression of inducible type I interferons ^[12]. In HCT116 ATG16L1 A300 cells, the basal production of type I interferons also was enhanced, and the cells had an increased sensitivity to the double stranded RNA mimic poly (I:C) via a mitochondrial anti-viral signaling (MAVS) pathway ^[21]. Zika vims down-regulates ATG16L1 expression and for Sindbis virus loss of ATG16L1 expression is associated with reduced eIF2a phosphorylation and viral protein synthesis ^{[29] [30]}. The anti-viral activity of interferon g also required ATG16L1 expression, with the ATG5-ATG12/ATG16L1 complex being required for norovirus to form its replication complex ^[31]. EXAMPLE 2

Ricin is a carbohydrate binding protein having two polypeptides. It is a type 2 ribosomal inactivating protein (RIP). Type 2 RIPs are composed of an A chain that is functionally equivalent to a type 1 RIP, covalently connected by a single disulfide bond to a B chain that is catalytically inactive, but serves to mediate transport of the A-B protein complex from the cell surface, via vesicle carriers, to the lumen of the endoplasmic reticulum (ER). Ricin then inactivates ribosomes. Within the lumen of the ER the ricin pro-polypeptide is glycosylated and protein disulfide isomerase (PDI) catalyzes disulfide bond formation between cysteines 294 and 318.

For ricin to function cytotoxically, the A peptide must initially be cleaved from the B peptide in order to release a steric inhibition of the A peptide active site which is catalyzed by PDI.

The combinations of [OSU-03012 + sildenafil] and of [sorafenib + sildenafil] were both demonstrated reduce the expression of PDI (aka HSP56) in GBM cells.

The A peptide in the ER lumen partially unfolds and partially buries into the ER membrane, where it is thought to mimic a misfolded membrane- associated protein. Roles for the ER localized-chaperones GRP94 and GRP78 have been proposed prior to the 'dislocation' of the A peptide from the ER lumen to the cytosol in a manner that utilizes components of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. ERAD normally removes misfolded ER proteins to the cytosol for their destruction by cytosolic proteasomes. Under normal circumstances, dislocation of the A peptide requires ER membrane- integral E3 ubiquitin ligase complexes but the A peptide avoids the ubiquitination that usually occurs with ERAD substrates because of its low content of lysine residues. Hence, under normal circumstances the A peptide avoids the usual fate of dislocated proteins (destruction by targeting ubiquitinylated proteins to the cytosolic proteasomes). In the mammalian cell cytosol, the A peptide then undergoes triage by the cytosolic molecular chaperones Hsc70 and Hsp90 and their co-chaperones, as well as by one subunit (RPT5) of the proteasome itself, that results in its folding to a catalytic conformation, which de-purinates ribosomes, thus halting all protein synthesis (thus causing death).

The combinations of [OSU-03012 + sildenafil] and of [sorafenib + sildenafil] were both demonstrated to potently inhibit the chaperone functions of GRP78 and GRP94 in the ER and cause a profound ER stress response concomitant with a rapid formation of autophagosomes. The activities of cytosolic HSP90 family chaperones and cytosolic HSP70 family chaperones are also inhibited by the drug combinations resulting in a further super- enhanced ER stress response with rapid autophagosome formation. Thus, ER-localized proteins after exposure to either drug combination are very rapidly formed into autophagosomes rapidly leading to autophagic flux and proteolytic digestion in autolysosomes i.e. there is no need for ubiquitination to cause rapid protein (A peptide) degradation.

Thus, the drug combinations block the actions of ricin at multiple stages of ricin processing, stages that are all required to deliver the active “killing” peptide:

(1) the combinations reduce the expression of PDI which prevents the initial cleavage of the A peptide and B peptide.

(2) the combinations inactivate GRP78 within the ER which causes autophagic flux to occur.

(3) the combinations inactivate multiple HSP90 family and HSP70 family chaperones so that the active A peptide cannot refold in the cytosol, which also further promotes autophagic flux.

(4) digestion of ricin peptides proceeds via the rapid formation of autophagosomes and flux to autolysosomes and does not require any ubiquitination step.

EXAMPLE 3

Bacteria express a homologue of GRP78, Dna K. Treatment of antibiotic sensitive wild type E. coli bacteria with AR-12 significantly reduced proliferation and enhanced penicillin / streptomycin toxicity. Treatment of laboratory generated ampicillin and kanamycin resistant E. coli bacteria with AR-12 significantly reduced proliferation. The present Example demonstrates that AR-12 down-regulates DNA K expression.

Bacteria also contain di-cyclic GMP phosphodiesterases that limit intracellular concentrations of cyclic nucleotides which when highly elevated are toxic. Thus, of particular note was that antibiotic resistant E. coli were also killed by the PDE5 inhibitors sildenafil (Viagra) or tadalafil (Cialis). In dose-response studies AR-12, sildenafil or tadalafil profoundly suppressed bacterial growth at very low clinically relevant concentrations. The decline in Dna K expression correlated with reduced expression of a Dna K chaperoned protein, Rec A. In the case of the chaperones GrpE (HSP27) and DNA J (HSP40), treatment with either sildenafil, tadalafil or AR-12 caused the appearance of a slower migrating species of the protein on SDS PAGE, that in mammalian cells would be linked to increased protein phosphorylation. These findings with OSU-03012 also correlated with significant morphological changes in the coliform bacteria, with surviving Gram-stained E. coli cells treated with AR-12 (2 mM) for 3 h appearing either elongated or “fat” in appearance.

Similar data to that obtained in E. coli with AR-12 were also found Neisseria gonorrhoeae strains FA19, FA1090, MS11 and 1291. AR-12 treatment did not appear to alter the morphology of these coccoid bacteria, though as before, we noted that AR-12 reduced expression of Dna K and Rec A. In FA19, AR-12 and PDE5 inhibitors could interact to cause a greater than additive amount of bacteria killing. Data in prior panels had shown that unlike the growth inhibitory antibiotic chloramphenicol, AR-12 exhibited true bacteriocidal effects on cells that had yet to enter log-phase growth.

Using F89 and H041 drug resistant N. gonorrhoeae isolates we determined whether OSU-03012 exhibited any antibiotic effect and whether it could enhance/restore the bacteriocidal properties of approved standard of care antibiotics. Treatment of F89 and H041 bacteria with AR-12 caused a dose-dependent reduction in bacterial growth. Treatment of F89 and H041 bacteria with AR-12 variably enhanced the lethality of Ceftriaxone, Ciprofloxin, or Azithromycin over a 5 h time course. Methicillin-resistant Staphylococcus epidermidis (MRSE) bacteria were noted to be at least as sensitive to AR-12 as were N. gonorrhoeae (F89, H041), possibly moreso i.e. 2 mM AR-12 was not growth inhibitory in F89 and H041 but reduced MRSA growth by ~50%, and co-exposure of bacteria to AR-12 and sildenafil modestly enhanced the cyto-toxic effect beyond that of AR-12 alone.

Sorafenib also exhibited antibiotic growth-suppressing properties in: Salmonella typhimurium and Streptococcus pyogenes with no activity against Vibrio cholerae. Higher concentrations of AR-12 than used for other bacteria studies were required to suppress Vibrio cholerae growth. We discovered that sorafenib could down-regulate expression of the bacterial GRP78-like chaperone Dna K, which was similar to our prior data using AR-12. The decline in Dna K expression correlated with reduced expression of a Dna K chaperoned protein, Rec A. In the case of the chaperone Dna J (HSP40), treatment with sorafenib also lowered its expression. Of greater significance in multi-drug antibiotic resistant forms of VRE; MRSA, MRSE, and antibiotic resistant Acinetobacter baumannii, sorafenib exhibited growth suppressive antibiotic properties as a single agent at low clinically relevant concentrations and in all except MRSE reverted antibiotic resistance to ampicillin, meropenem or vancomycin. The bacterial growth suppressive effect of sorafenib on MRSE is of interest because in a very small subset of cancer patients sorafenib can cause a facial acneiform eruption, that could be Staphylococcus epidermis -related.

Generic laboratory strain Klebsiella pneumoniae is inherently resistant to ampicillin, and we additionally obtained multiple strains of this bacterial species from the VCU-MCVH bacterial pathology laboratory and determined the impact of sorafenib with or without antibiotic exposure on their growth (Fig. 13 A-C). Sorafenib, AR- 12 or pazopanib significantly reduced the growth of “generic” laboratory strain Klebsiella pneumoniae. In carbapenem- resistant Klebsiella pneumoniae strain #1 over-expressing very high levels of the resistance enzyme bla kpc, bacterial growth was significantly inhibited by either sorafenib or pazopanib; and sorafenib significantly enhanced the antibiotic properties of ampicillin but for unknown reasons surprisingly not of meropenem.

Antibiotics can have multiple effects on the morphology of bacteria, in particular as we previously observed for the Dna K inhibitory chemical AR-12, on coliform morphology where rod shaped bacteria either became coccoid in appearance or alternatively bacteria became ~4x longer and had ~2x more width. Drug-treated bacteria were smeared onto glass slides, fixed and Gram stained. Klebsiella pneumoniae bacteria of all three strains under control conditions displayed a short fat rod shaped appearance with the length of the rod being approximately twice the width. Growth of all Klebsiella strains in the presence of 5 mM sorafenib resulted in bacteria which appeared coccoid in shape, even going so far as to form long chains of cells, reminiscent of Rouleux chains of clotting red blood cells. A very few bacteria, in strain #4, appeared to remain in large rod-like structures after sorafenib exposure but that were less intensely stained that in vehicle control treated bacteria. Collectively, these data show that sorafenib, possibly by altering Dna K function, is altering the biology of the Klebsiella pneumoniae cell membrane.

Althogether, AR-12, sorafenib tosylate and PDE5 inhibitors may be used to suppress the growth of Bacillus Anthracis as well as other potential bio-weapon bacteria such as Botulinum and Yersinia Pestis.

References for Example 1

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6. Booth L., Roberts J.L., Tavallai M., Nourbakhsh A., Chuckalovcak J., Carter J., Poklepovic A., Dent P. OSU-03012 and viagra treatment inhibits the activity of multiple chaperone proteins and disrupts the blood-brain barrier: implications for anti-cancer therapies: AR-12 AND VIAGRA. J. Cell. Physiol. 2015;230(8): 1982-1998.

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20. Sadaghian Sadabad M., Regeling A., de Goffau M.C., Blokzijl T., Weersma R.K., Penders L, Faber K.N., Harmsen H.J.M., Dijkstra G. The ATG16L1-T300A allele impairs clearance of pathosymbionts in the inflamed ileal mucosa of Crohn's disease patients. Gut. 2015 ;64(10): 1546-1552.

21. Lassen K.G., Kuballa P., Conway K.L., Patel K.K., Becker C.E., Peloquin J.M., Villablanca E.J., Norman J.M., Liu T.-C., Heath R.J., Becker M.L., Fagbami L., Horn H., Mercer L, Yilmaz O.H., Jaffe J.D., Shamji A.F., Bhan A.K., Carr S.A., Daly M.J., Virgin H.W., Schreiber S.L., Stappenbeck T.S., Xavier R.J. Atgl6Ll T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense. Proc. Natl. Acad. Sci. 2014;lll(21):7741-7746.

22. Li Q.-X., Zhou X., Huang T.-T., Tang Y., Liu B.o., Peng P., Sun L.i., Wang Y.-H., Yuan X.-L. The Thr300Ala variant of ATG16L1 is associated with decreased risk of brain metastasis in patients with non-small cell lung cancer. Autophagy. 2017;13(6):1053-1063.

23. Shulla A., Heald-Sargent T., Subramanya G., Zhao L, Perlman S., Gallagher T. A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry. J. Virol. 2011;85:873-882.

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25. Renteria A.E., Endam Mfuna L., Adam D., Filali-Mouhim A., Maniakas A., Rousseau S., Brochiero E., Gallo S., Desrosiers M. Azithromycin downregulates gene expression of il- 1b and pathways involving TMPRSS2 and TMPRSS 1 ID required by SARS-CoV-2. Am J Respir Cell Mol Biol. 2020 Aug 28 doi: 10.1165/rcmb.2020-0285LE.

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28. Zeh H.J., Bahary N., Boone B.A., Singhi A.D., Miller-Ocuin J.L., Normolle D.P., Zureikat A.H., Hogg M.E., Bartlett D.L., Lee K.K., Tsung A., Marsh J.W., Murthy P., Tang D., Seiser N., Amaravadi R.K., Espina V., Liotta L., Lotze M.T. A Randomized Phase II Preoperative Study of Autophagy Inhibition with High-Dose Hydroxychloroquine and Gemcitabine/Nab-Paclitaxel in Pancreatic Cancer Patients. Clin Cancer Res. 2020;26

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References for Example 2

Spooner RA, Watson PD, Marsden CJ, Smith DC, Moore KA, Cook JP, Lord JM, Roberts LM (October 2004). "Protein disulphide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum". Biochem. J. 383 (Pt 2): 285-93. doi:10.1042/BJ20040742. PMC 1134069. PMID 15225124.

Bellisola G, Fracasso G, Ippoliti R, Menestrina G, Rosen A, Solda S, Udali S, Tomazzolli R, Tridente G, Colombatti M (May 2004). "Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase". Biochem. Pharmacol. 67 (9): 1721-31. doi:10.1016/j.bcp.2004.01.013. PMID 15081871. Spooner RA, Hart PJ, Cook JP, Pietroni P, Rogon C, Hohfeld J, Roberts LM, Lord JM (November 2008). "Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum". Proc. Natl. Acad. Sci. U.S.A. 105 (45): 17408-13. Slominska-Wojewodzka M, Gregers TF, Walchli S, Sandvig K (April 2006). "EDEM is involved in retrotranslocation of ricin from the endoplasmic reticulum to the cytosol". Mol. Biol. Cell. 17 (4): 1664-75. doi:10.1091/mbc.E05- 10-0961. PMC 1415288. PMID 16452630. Li S, Spooner RA, Allen SC, Guise CP, Ladds G, Schnoder T, Schmitt MJ, Lord JM, Roberts LM (August 2010). "Folding-competent and folding-defective forms of ricin A chain have different fates after retrotranslocation from the endoplasmic reticulum". Mol. Biol. Cell. 21 (15): 2543-54. doi:10.1091/mbc.E09-08-0743. PMC 2912342. PMID 20519439.

Deeks ED, Cook JP, Day PJ, Smith DC, Roberts LM, Lord JM (March 2002). "The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol". Biochemistry. 41 (10): 3405-13. doi:10.1021/bi011580v. PMID 11876649.

While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

CLAIMS We claim:

1. A method of treating a coronavirus infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising at least one compound or pharmaceutically acceptable salt, solvate or hydrate thereof having the general chemical structure of:

wherein Xi is H, or an unbranched, branched, saturated, unsaturated, substituted, or unsubstituted C1-C8 alkyl group; and

X2 is an unbranched, branched, saturated, unsaturated, substituted, or unsubstituted C1-C8 alkylcarbonyl; substituted or unsubstituted aryl or heteroarylcarbonyl; substituted or unsubstituted alkylsulfonyl; carboxamide, sulfonamide, aminosulfonamide, acylamido, or H.

2. The method of claim 1, wherein the coronavirus is SARS-CoV-2.

3. The method of claim 1, wherein the at least one compound is AR-12.

4. The method of claim 1, wherein the at least one compound is AR-13.

5. The method of claim 1, further comprising administering one or more PDE5 inhibitors.

6. The method of claim 3, wherein the one or more PDE5 inhibitors is selected from the group consisting of sildenafil, vardenafil, tadalafil, avanafil, udenafil, microdenafil, and lodenafil carbonate.

7. A method of treating a coronavirus infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising sorafenib tosylate or pazopanib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

8. The method of claim 7, wherein the coronavirus is SARS-CoV-2.

9. The method of claim 7, further comprising administering one or more PDE5 inhibitors.

10. The method of claim 9, wherein the one or more PDE5 inhibitors is selected from the group consisting of sildenafil, vardenafil, tadalafil, avanafil, udenafil, microdenafil, and lodenafil carbonate.

11. A method of treating ricin poisioning in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising at least one compound or pharmaceutically acceptable salt, solvate or hydrate thereof having the general chemical structure of:

wherein Xi is H, or an unbranched, branched, saturated, unsaturated, substituted, or unsubstituted C1-C8 alkyl group; and

X2 is an unbranched, branched, saturated, unsaturated, substituted, or unsubstituted C1-C8 alkylcarbonyl; substituted or unsubstituted aryl or heteroarylcarbonyl; substituted or unsubstituted alkylsulfonyl; carboxamide, sulfonamide, aminosulfonamide, acylamido, or H.

12. The method of claim 11, wherein the at least one compound is AR-12.

13. The method of claim 11, wherein the at least one compound is AR-13.

14. The method of claim 11, further comprising administering one or more PDE5 inhibitors.

15. The method of claim 14, wherein the one or more PDE5 inhibitors is selected from the group consisting of sildenafil, vardenafil, tadalafil, avanafil, udenafil, microdenafil, and lodenafil carbonate.

16. A method of treating ricin poisioning in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising sorafenib tosylate or pazopanib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

17. The method of claim 15, further comprising administering one or more PDE5 inhibitors.

18. The method of claim 17, wherein the one or more PDE5 inhibitors is selected from the group consisting of sildenafil, vardenafil, tadalafil, avanafil, udenafil, microdenafil, and lodenafil carbonate.

19. A method of inhibiting the growth of rod-shaped bacteria, comprising contacting the rod-shaped bacteria with a composition comprising at least one compound or pharmaceutically acceptable salt, solvate or hydrate thereof having the general chemical structure of: wherein

Xi is H, or an unbranched, branched, saturated, unsaturated, substituted, or unsubstituted C1-C8 alkyl group; and

X2 is an unbranched, branched, saturated, unsaturated, substituted, or unsubstituted C1-C8 alkylcarbonyl; substituted or unsubstituted aryl or heteroarylcarbonyl; substituted or unsubstituted alkylsulfonyl; carboxamide, sulfonamide, aminosulfonamide, acylamido, or H.

20. The method of claim 19, wherein the at least one compound is AR-12.

21. The method of claim 19, wherein the at least one compound is AR-13.

22. The method of claim 19, wherein the rod-shaped bacteria is selected from the group consisting of Bacillus, Clostridium, Yersinia, and Klebsiella.

23. The method of claim 19, further comprising contacting the rod-shaped bacteria with one or more PDE5 inhibitors.

24. The method of claim 23, wherein the one or more PDE5 inhibitors is selected from the group consisting of sildenafil, vardenafil, tadalafil, avanafil, udenafil, microdenafil, and lodenafil carbonate.

25. A method of inhibiting the growth of rod-shaped bacteria, comprising contacting the rod-shaped bacteria with sorafenib tosylate or pazopanib or a pharmaceutically acceptable salt, solvate or hydrate thereof.

26. The method of claim 25, wherein the rod-shaped bacteria is selected from the group consisting of Bacillus, Clostridium, Yersinia, and Klebsiella.

27. The method of claim 25, further comprising contacting the rod-shaped bacteria with one or more PDE5 inhibitors.

28. The method of claim 27, wherein the one or more PDE5 inhibitors is selected from the group consisting of sildenafil, vardenafil, tadalafil, avanafil, udenafil, microdenafil, and lodenafil carbonate.