# EDGAR Filing Document

**Accession Number:** 0001430306
**File Stem:** 0001387131-23-000747
**Filing Date:** 2023-1
**Character Count:** 32158
**Document Hash:** 18e6d782f2dcaadb9076f949ff36a06c
**Contains OCR:** False
**Source Format:** 

## Filing Content

## Filing Summary
**0001387131-23-000747.hdr.sgml**: 20230126

**ACCESSION NUMBER**: 0001387131-23-000747

**CONFORMED SUBMISSION TYPE**: 8-K

**PUBLIC DOCUMENT COUNT**: 50

**CONFORMED PERIOD OF REPORT**: 20230126

**ITEM INFORMATION**: Regulation FD Disclosure

**ITEM INFORMATION**: Other Events

**ITEM INFORMATION**: Financial Statements and Exhibits

**FILED AS OF DATE**: 20230126

**DATE AS OF CHANGE**: 20230126

**FILER**: 

**COMPANY DATA:**
- **COMPANY CONFORMED NAME:** Tonix Pharmaceuticals Holding Corp.
- **CENTRAL INDEX KEY:** 0001430306
- **STANDARD INDUSTRIAL CLASSIFICATION:** PHARMACEUTICAL PREPARATIONS [2834]
- **IRS NUMBER:** 261434750
- **STATE OF INCORPORATION:** NV
- **FISCAL YEAR END:** 1231

**FILING VALUES:**
- **FORM TYPE:** 8-K
- **SEC ACT:** 1934 Act
- **SEC FILE NUMBER:** 001-36019
- **FILM NUMBER:** 23558620

**BUSINESS ADDRESS:**
- **STREET 1:** 26 MAIN STREET, SUITE 101
- **CITY:** CHATHAM
- **STATE:** NJ
- **ZIP:** 07928
- **BUSINESS PHONE:** 212-980-9155

**MAIL ADDRESS:**
- **STREET 1:** 26 MAIN STREET, SUITE 101
- **CITY:** CHATHAM
- **STATE:** NJ
- **ZIP:** 07928

**FORMER COMPANY:**
- **FORMER CONFORMED NAME:** TAMANDARE EXPLORATIONS INC.
- **DATE OF NAME CHANGE:** 20080320

?xml version="1.0" encoding="utf-8"?

**UNITED STATES**

**SECURITIES AND EXCHANGE COMMISSION**

**Washington, D.C. 20549**

**FORM 8-K**

**CURRENT REPORT**

**Pursuant to Section 13 or 15(d) of the Securities Exchange Act of 1934**

**Date of report (date of earliest event reported): January 26, 2023**

**TONIX PHARMACEUTICALS HOLDING CORP.**

**(Exact name of registrant as specified in its charter)**

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| | | |
|:---|:---|:---|
| **Nevada** | **001-36019** | **26-1434750** |
| **(State or Other Jurisdiction** <br> **of Incorporation)**  | **(Commission** <br> **File Number)** | **(IRS Employer** <br> **Identification No.)**  |

---

26 Main Street, Chatham, New Jersey 07928

**(Address of principal executive offices) (Zip Code)**

**Registrant's telephone number, including area code:** (862) 904-8182

Check the appropriate box below if the Form 8-K filing is intended to simultaneously satisfy the filing obligation of the registrant under any of the following provisions (see General Instruction A.2. below):

☐ Written communications pursuant to Rule 425 under the Securities Act (17 CFR 230.425)

☐ Soliciting material pursuant to Rule 14a-12 under the Exchange Act (17 CFR 240.14a-12)

☐ Pre-commencement communications pursuant to Rule 14d-2(b) under the Exchange Act (17 CFR 240.14d-2(b))

☐ Pre-commencement communications pursuant to Rule 13e-4(c) under the Exchange Act (17 CFR 240.13e-4(c))

Securities registered pursuant to Section 12(b) of the Act:

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| | | |
|:---|:---|:---|
| Title of each class | Trading Symbol(s) | Name of each exchange on which registered |
| Common Stock | TNXP | The NASDAQ Capital Market |

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Indicate by check mark whether the registrant is an emerging growth company as defined in Rule 405 of the Securities Act of 1933 (§ 230.405 of this chapter) or Rule 12b-2 of the Securities Exchange Act of 1934 (§ 240.12b-2 of this chapter).

Emerging growth company ☐

If an emerging growth company, indicate by check mark if the registrant has elected not to use the extended transition period for complying with any new or revised financial accounting standards provided pursuant to Section 13(a) of the Exchange Act. ☐

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| | |
|:---|:---|
| **Item 7.01** | **&nbsp;&nbsp;&nbsp;&nbsp; Regulation FD Disclosure.** |

---

On January 26, 2023, Tonix Pharmaceuticals Holding Corp. (the "Company") announced that Tom Hobman, Ph.D., Professor of Cell Biology, University of Alberta, presented data from his laboratory at The University of Alberta during an oral presentation at the 2nd Wnt & β-Catenin Targeted Drug Development Conference on January 26, 2023 (the "Presentation"). A copy of the press release which discusses this matter is furnished hereto as Exhibit 99.01, and incorporated herein by reference. The Presentation, which may contain nonpublic information, is filed as Exhibit 99.02 hereto and incorporated herein by reference.

The information in this Item 7.01 of this Current Report on Form 8-K, including Exhibits 99.01 and 99.02 attached hereto, shall not be deemed "filed" for purposes of Section 18 of the United States Securities Exchange Act of 1934 (the "Exchange Act") or otherwise subject to the liabilities of that section, nor shall they be deemed incorporated by reference in any filing under the United States Securities Act of 1933 or the Exchange Act, except as shall be expressly set forth by specific reference in such a filing.

---

| | |
|:---|:---|
| **Item 8.01.** | **Other Events.** |

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On January 26, 2023, the Company announced that Dr. Hobman presented data from the Presentation, entitled "*Targeting the Wnt/β-catenin pathway as a broad-spectrum antiviral strategy*," which includes research sponsored by the Company focused on the development and testing of Wnt/β-catenin signaling pathway inhibitors as broad-spectrum antivirals against SARS-CoV-2 and other emerging viruses. The research demonstrated that inhibition of Wnt/β-catenin pathway induces peroxisomes and enhances interferon response during viral infection, significantly reducing SARS-CoV-2 replication *in vitro* and *in vivo* The Company previously announced that it exercised an option to license the antiviral technology platform.

*Forward- Looking Statements*

This Current Report on Form 8-K contains certain forward-looking statements within the meaning of Section 27A of the Securities Act of 1933 and Section 21E of the Securities Exchange Act of 1934 and Private Securities Litigation Reform Act, as amended, including those relating to the Company's product development, clinical trials, clinical and regulatory timelines, market opportunity, competitive position, possible or assumed future results of operations, business strategies, potential growth opportunities and other statement that are predictive in nature. These forward-looking statements are based on current expectations, estimates, forecasts and projections about the industry and markets in which we operate and management's current beliefs and assumptions.

These statements may be identified by the use of forward-looking expressions, including, but not limited to, "expect," "anticipate," "intend," "plan," "believe," "estimate," "potential," "predict," "project," "should," "would" and similar expressions and the negatives of those terms. These statements relate to future events or our financial performance and involve known and unknown risks, uncertainties, and other factors which may cause actual results, performance or achievements to be materially different from any future results, performance or achievements expressed or implied by the forward-looking statements. Such factors include those set forth in the Company's filings with the SEC. Prospective investors are cautioned not to place undue reliance on such forward-looking statements, which speak only as of the date of this press release. The Company undertakes no obligation to publicly update any forward-looking statement, whether as a result of new information, future events or otherwise.

---

| | |
|:---|:---|
| **Item 9.01** | **Financial Statements and Exhibits.** |

---

---

| | | |
|:---|:---|:---|
| (d) | **Exhibit**<br> **No.** | **Description** |
|  | [99.01](ex99-01.htm)<br> [99.02](ex99-02.htm)<br> 104 | Press release of the Company, dated January 26, 2023<br> Targeting the Wnt/β-catenin pathway as a broad-spectrum antiviral strategy<br> Cover Page Interactive Data File (embedded within the Inline XBRL document) |

---

**SIGNATURE**

Pursuant to the requirement of the Securities Exchange Act of 1934, the registrant has duly caused this report to be signed on its behalf by the undersigned thereunto duly authorized.

---

| | | |
|:---|:---|:---|
|  | **TONIX PHARMACEUTICALS HOLDING CORP.** | **TONIX PHARMACEUTICALS HOLDING CORP.** |
| Date: January 26, 2023 | By: | /s/ Bradley Saenger |
|  | Bradley Saenger | Bradley Saenger |
|  | Chief Financial Officer | Chief Financial Officer |

---

## Exhibit 99.01

[TONIX PHARMACEUTICALS HOLDING CORP. FORM 8-K](tnxp_8k-012623.htm)

**Exhibit 99.01**

**Tonix Pharmaceuticals Announces Presentation of Licensed Antiviral Drug Technology at the 2<sup>nd</sup> Wnt & β-catenin Targeted Drug Development Conference**

 

*Oral Presentation Describes Activity of Wnt/β-catenin Signaling Pathway Inhibitors Against SARS-CoV-2 in Cell Culture and in an Animal Model*

 

CHATHAM, N.J., January 26, 2023 – Tonix Pharmaceuticals Holding Corp. (Nasdaq: TNXP) (Tonix or the Company), a clinical-stage biopharmaceutical company, today announced that Tom Hobman, Ph.D., Professor of Cell Biology, University of Alberta, presented data from his laboratory at The University of Alberta during a presentation at the 2<sup>nd</sup> Wnt & β-catenin Targeted Drug Development Conference held in Boston, Mass., on January 26, 2023. The oral presentation titled, "***Targeting the Wnt/β -catenin pathway as a broad-spectrum antiviral strategy***," includes research sponsored by Tonix Pharmaceuticals focused on the development and testing of Wnt/β-catenin signaling pathway inhibitors as broad-spectrum antivirals against SARS-CoV-2 and other emerging viruses. Tonix has previously announced that it exercised an option to license the antiviral technology platform. A copy of the presentation is available on the Tonix Pharmaceuticals corporate website at <u>www.tonixpharma.com</u>.

"Antiviral therapeutics are needed to mitigate the effects of SARS-CoV-2 and future coronavirus outbreaks, and Professor Hobman's work is designed to facilitate the identification and testing of novel broad-spectrum antiviral drugs," said Seth Lederman, M.D., Chief Executive Officer of Tonix Pharmaceuticals. "Professor Hobman presented data showing that inhibition of Wnt/β-catenin pathway induces peroxisomes and enhances interferon response during viral infection, significantly reducing SARS-CoV-2 replication *in vitro* and *in vivo*."

"For future pandemics, the scientific community must be ready with an arsenal of easily self-administered drugs that can be tested in rapid, efficient clinical trials immediately after the causative viral agent is identified," said Professor Tom Hobman. "The research collaboration between Tonix and The University of Alberta is focused on the development and testing of Wnt/β-catenin signaling pathway inhibitors as broad-spectrum antivirals against SARS-CoV-2 and other emerging viruses."

**Tonix Pharmaceuticals Holding Corp.<sup>\*</sup>**

Tonix is a clinical-stage biopharmaceutical company focused on discovering, licensing, acquiring and developing therapeutics to treat and prevent human disease and alleviate suffering. Tonix's portfolio is composed of central nervous system (CNS), rare disease, immunology and infectious disease product candidates. Tonix's CNS portfolio includes both small molecules and biologics to treat pain, neurologic, psychiatric and addiction conditions. Tonix's lead CNS candidate, TNX-102 SL (cyclobenzaprine HCl sublingual tablet), is in mid-Phase 3 development for the management of fibromyalgia with a new Phase 3 study launched in the second quarter of 2022 and interim data expected in the second quarter of 2023. TNX-102 SL is also being developed to treat Long COVID, a chronic post-acute COVID-19 condition. Tonix initiated a Phase 2 study in Long COVID in the third quarter of 2022 and expects interim data in the third quarter of 2023. TNX-1300 (cocaine esterase) is a biologic designed to treat cocaine intoxication and has been granted Breakthrough Therapy designation by the FDA. A Phase 2 study of TNX-1300 is expected to be initiated in the first quarter of 2023. TNX-1900 (intranasal potentiated oxytocin), a small molecule in development for chronic migraine, is expected to enter the clinic with a Phase 2 study in the first quarter of 2023. TNX-601 ER (tianeptine hemioxalate extended-release tablets) is a once-daily formulation of tianeptine being developed as a potential treatment for major depressive disorder (MDD) with a Phase 2 study expected to be initiated in the first quarter of 2023. Tonix's rare disease portfolio includes TNX-2900 (intranasal potentiated oxytocin) for the treatment of Prader-Willi syndrome. TNX-2900 has been granted Orphan Drug designation by the FDA. Tonix's immunology portfolio includes biologics to address organ transplant rejection, autoimmunity and cancer, including TNX-1500, which is a humanized monoclonal antibody targeting CD40-ligand (CD40L or CD154) being developed for the prevention of allograft and xenograft rejection and for the treatment of autoimmune diseases. A Phase 1 study of TNX-1500 is expected to be initiated in the first half of 2023. Tonix's infectious disease pipeline includes a vaccine in development to prevent smallpox and monkeypox, TNX-801, a next-generation vaccine to prevent COVID-19, TNX-1850, a platform to make fully human monoclonal antibodies to treat COVID-19, TNX-3600, and humanized anti-SARS-CoV-2 monoclonal antibodies, TNX-3800, recently licensed from Curia. TNX-801, Tonix's vaccine in development to prevent smallpox and monkeypox, also serves as the live virus vaccine platform or recombinant pox vaccine (RPV) platform for other infectious diseases. A Phase 1 study of TNX-801 is expected to be initiated in Kenya in the second half of 2023.

*<sup>\*</sup>All of Tonix's product candidates are investigational new drugs or biologics and have not been approved for any indication.*

This press release and further information about Tonix can be found at <u>www.tonixpharma.com</u>.

**Forward Looking Statements**

Certain statements in this press release are forward-looking within the meaning of the Private Securities Litigation Reform Act of 1995. These statements may be identified by the use of forward-looking words such as "anticipate," "believe," "forecast," "estimate," "expect," and "intend," among others. These forward-looking statements are based on Tonix's current expectations and actual results could differ materially. There are a number of factors that could cause actual events to differ materially from those indicated by such forward-looking statements. These factors include, but are not limited to, risks related to the failure to obtain FDA clearances or approvals and noncompliance with FDA regulations; delays and uncertainties caused by the global COVID-19 pandemic; risks related to the timing and progress of clinical development of our product candidates; our need for additional financing; uncertainties of patent protection and litigation; uncertainties of government or third party payor reimbursement; limited research and development efforts and dependence upon third parties; and substantial competition. As with any pharmaceutical under development, there are significant risks in the development, regulatory approval and commercialization of new products. Tonix does not undertake an obligation to update or revise any forward-looking statement. Investors should read the risk factors set forth in the Annual Report on Form 10-K for the year ended December 31, 2021, as filed with the Securities and Exchange Commission (the "SEC") on March 14, 2022, and periodic reports filed with the SEC on or after the date thereof. All of Tonix's forward-looking statements are expressly qualified by all such risk factors and other cautionary statements. The information set forth herein speaks only as of the date thereof.

**Contacts**

Jessica Morris (corporate)

Tonix Pharmaceuticals

investor.relations@tonixpharma.com

(862) 904-8182

Olipriya Das, Ph.D. (media)

Russo Partners

Olipriya.Das@russopartnersllc.com

(646) 942-5588

Peter Vozzo (investors)

ICR Westwicke

peter.vozzo@westwicke.com

(443) 213-0505

## Exhibit 99.02

[TONIX PHARMACEUTICALS HOLDING CORP. FORM 8-K](tnxp_8k-012623.htm)

**Exhibit 99.02**

![](ex9902img001.jpg)

Targeting the Wnt/b-catenin pathway as a broad-spectrum antiviral strategy Tom C. Hobman Department of Cell Biology Li Ka Shing Institute of Virology University of Alberta

![](ex9902img002.jpg)

Disclosures • Received research funding from Tonix Pharmaceuticals • Licensing agreement with Tonix Pharmaceuticals

![](ex9902img003.jpg)

Researchapproaches Focus Experimental § •Identification Flaviviruses of key host factors/pathways that •are Alphaviruses utilized or affected by multiple RNA viruses • HIV § •Pharmacological Coronaviruses • PR S I CR targeting of these host Restriction & Dependency factors/pathways should Factors result in broad-spectrum Determine cellular pathways are perturbed or activated antiviralwhich activity • Test effects of pathway agonists/inhibitors on virus replication

![](ex9902img004.jpg)

"For the present pandemic response, and for future pandemics …. the scientific community must be ready with an arsenal of easily self-administered drugs that can be tested in rapid, efficient clinical trials immediately after the causative viral agent is identified." White et al, 2021 Host-targeted antivirals to be part of this arsenal?

![](ex9902img005.jpg)

Peroxisomes are targeted during RNA virus infection • Abundant metabolic organelles in the cytoplasm • Catabolize very long chain fatty acids • Regulate reactive oxygen species • Produce specialized phospholipids (e.g. plasmalogens)

![](ex9902img006.jpg)

Flavivirus infection results in loss of peroxisomes Mock WNV Mock WNV Peroxisome aNS2B-3 Peroxisome You, Hou et al. 2015

![](ex9902img007.jpg)

Why would a virus want to deplete the Trends in Cell Biology peroxisome pool? Ferreira et al, 2022 Trends in Cell Biology Figure 1. Mitochondrial antiviral signaling (MAVS) protein-dependent antiviral signaling. Upon virus entry, viral RNA is released into the cytosol where it is recognized by retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5). This recognition allows the activation of MAVS at both mitochondria and peroxisomes. MAVS then undergoes a conformational change that allows its oligomerization, which is essential for the activation of a signaling cascade that activates the transcription factors interferon regulatory factors IRF1 and IRF3. Translocation of IRF1 and IRF3 to the nucleus promotes the expression of type I and type III interferons (IFNs) and IFN-stimulated genes (ISGs). Figure prepared using BioRender.com. >>Peroxisomes are antiviral signaling platforms that facilitate induction of type I and III interferons (IFN) inhibits downstream signaling [35] (Figure 2). Although vMIA also inhibits mitochondria-dependent MAVS signaling, HCMV seems to have developed distinct mechanisms to interfere with antiviral signaling at both organelles. In fact, although vMIA inhibits mitochondria-dependent signaling

![](ex9902img008.jpg)

SARS-CoV-2 is highly sensitive to Interferon (IFN) SARS-CoV-2 Is Sensitive to Type I IFN Pretreatment Lokugamage et al. FIG 1 SARS-CoV-2 is sensitive to type I IFN pretreatment. (A) Vero E6 cells were treated with 1,000 U recombinant type I (hashed line) IFN or mock (solid line) for 18 h prior to infection. The cells were subseque infected with either SARS-CoV wild type (WT; black) or SARS-CoV-2 (blue) at an MOI of 0.01, as described in the Each point on the line graph represents the group mean (n # 3). All error bars represent the standard deviat (SD). A two-tailed Student t test was used to determine P values (\*\*\*, P ! 0.001). (B) Vero E6 cell protein lysates Lokugamage et al, 2020 IFN-I-treated and untreated cells were probed at 48 h postinfection by Western blotting for phosphorylated ST (Y701), STAT1, IFITM1, SARS spike, and actin.

![](ex9902img009.jpg)

SARS-CoV-2 depletes functional peroxisomes SARS-CoV-2 24-hr 48-hr 72-hr a-PTS1 Mock Flavivirus Knoblach et al, 2021 X Fujiki et al, 2014 Mock 24 48 72 SARS-CoV-2 hpi

![](ex9902img010.jpg)

Respiratory Syncytial Virus (RSV) also Respiratory syncytial virus infection depletes reduces peroxisome pool peroxisomes too RSV Mock RSV F Peroxisomes Merge

![](ex9902img011.jpg)

As does HIV…. Xu et al, 2017

![](ex9902img012.jpg)

Patel et al, 2020

![](ex9902img013.jpg)

HIV-1 Vpu suppresses peroxisome biogenesis Vpu reduces metabolic activity of peroxisomes RESEARCH ARTICLE Host-Microbe Biology crossm RESEARCH ARTICLE Host-Microbe Biology crossm The HIV-1 Accessory Protein Vpu Downregulates Peroxisome Biogenesis The HIV-1 Accessory Protein Peroxisome Zaikun Xu, Robert Lodge, Christopher Power, Eric A.Vpu Cohen, Downregulates Tom C. Hobman Biogenesis Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada a b c,d,f,g b,h a,d,e,f a Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal, Montréal, Quebec, Canada b b c,d,f,g Eric A. Cohen,b,h Tom C. Hobmana,d,e,f Zaikun Xu,ofa Medicine, Robert Lodge, Power, Department University ofChristopher Alberta, Edmonton, Alberta, Canada c Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada bLaboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal, Montréal, Quebec, Canada fWomen & Children's Health Research Institute, University of Alberta, Edmonton, Alberta, Canada d a e Department Medicine, of Alberta, Edmonton, Alberta, Canada Alberta, Canada Neuroscienceofand MentalUniversity Health Institute, University of Alberta, Edmonton, Department of of Microbiology, Medical Microbiology and Immunology, University ofUniversité Alberta, Edmonton, Alberta, Canada Department Infectious Diseases and Immunology, de Montréal, Montréal, Quebec, Canada c g d h Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada e Women & Children's Healthimmunodeficiency Research Institute, University Alberta, Edmonton, Alberta, Canada ABSTRACT Human virus oftype 1 (HIV-1) establishes lifelong infecgNeuroscience and Mental Health Institute, University of Alberta, Edmonton, Alberta, Canada tions in humans, a process that relies on its ability to thwart innate and adaptive imf Department of Microbiology, Infectious Diseases and Université de Montréal, Montréal, mune defenses of the host. Recently, weImmunology, reported that HIV-1 infection resultsQuebec, in a Canada dramatic reduction of the cellular peroxisome pool. Peroxisomes are metabolic orABSTRACT immunodeficiency type 1 in(HIV-1) establishes lifelong infecganelles thatHuman also function as signalingvirus platforms the innate immune response. tions in humans, a process that relies on its ability to thwart innate and adaptive Here, we show that the HIV-1 accessory protein Vpu is necessary and sufficient imfor mune defensesofofcellular the host. Recently, during we reported that Vpu HIV-1induces infection in a the depletion peroxisomes infection. the results expression dramatic reduction that of thetarget cellular peroxisome pool. Peroxisomes orof four microRNAs mRNAs encoding proteins requiredareformetabolic peroxisome ganelles that also function as signaling platforms in the innate immune response. formation and metabolic function. The ability of Vpu to downregulate peroxisomes Here, we show the HIV-1upon accessory protein Vpu issignaling necessary and sufficient for was found to bethat dependent the Wnt/ !-catenin pathway. Given the the depletion cellular peroxisomes during infection. the expression importance of of peroxisomes in innate immune signaling Vpu and induces central nervous system :d0ea:d18c:d071:5b5b. 1:5b5b. h Hopfensperger et al, 2020

![](ex9902img014.jpg)

§ Wnt/b pathway inhibits peroxisome biogenesis Hypothesis: Inhibiting Wnt/b pathway will induce peroxisomes and reduce virus replication via enhanced IFN response Test effects of Wnt/b pathway inhibitors on peroxisome density

![](ex9902img015.jpg)

Wnt/β-catenin inhibitors increase peroxisome density DMSO KYA1797K Pyrvinium E7449 But do they inhibit virus replication? Xu, Elaish, Wong et al, in preparation

![](ex9902img016.jpg)

Wnt/β-catenin inhibitors reduce SARS-CoV-2 replication in multiple cell types Calu-3 Calu3 Calu3-human lung adenocarcinoma NHBE-normal human bronchial epithelial cells NHBE NHBE Xu, Elaish, Wong et al, in preparation

![](ex9902img017.jpg)

Reducing β-catenin levels induces peroxisome proliferation and enhances IFN response X Sawa et al, 2013 Suggests 38that antiviral effects of Wnt/b catenin inhibitors is not due to off target effects Xu, Elaish, Wong et al, in preparation

![](ex9902img018.jpg)

Wnt/β-catenin inhibitors increase peroxisome density in Vero cells but do not reduce virus replication 5 µM 1 µM 4 0 4 7 4 0 P y S M E 6 9 -1 T R iC 9 1 1 4 0 1 m in iu iu in rv µM n 0 0 1 0 m 1 9 5 1 2 rv y P M M n µM µM µM 0 2 9 1 E T K C Y -1 A A Y K 1 K 7 7 1 9 7 8 9 4 7 6 K 1 0 1 µM µM µM 1 6 -0 B B N C C N n t- -0 C 8 5 4 9 1 1 0 0 µM µM 1 4 W K L L G P K -9 7 -9 -O 7 1 4 1 0 1 1 IW G S M D -O P IW µM µM 4 O S A R S - C o V - 2 v ir u s tit e r (P F U /m l, L o g) V e ro E 6 , 2 4 h p i 6 Consistent with model that antiviral effects of these drugs are IFN-dependent Xu, Elaish, Wong et al, in preparation

![](ex9902img019.jpg)

T S M 2 E 0 7 9 K 4 4 9 -1 6 6 9 4 9 0 4 m 5 7 iu 1 9 T 4 R in 2 rv 9 7 4 5 7 1 O \* iC y -1 9 0 4 P C 8 C -0 t- 1 B A C Y N n -O S -9 P M K 4 9 5 W G 4 6 4 3 L 7 4 -1 4 S A R S - C o V - 2 v ir u s t it e r (P F U /m l, L o g) E 0 T \* D M R 9 K 6 m 5 7 iu 1 9 4 5 IW S 2 in 2 rv 9 7 8 9 \* iC y -1 1 -0 5 \* P C A B C 6 K T Y C 4 1 2 h p o s t- in fe c tio n tr e a tm e n t, 2 4 h p i E E K N t- 1 O 7 -O S -9 P M K n G W L IW D S A R S - C o V - 2 v ir u s t it e r (P F U /m l, L o g) Some Wnt/β-catenin inhibitors decrease virus replication when added post-infection 1 2 h p o s t- in fe c tio n tr e a tm e n t, 4 8 h p i 6 \* \* 3 Xu, Elaish, Wong et al, in preparation

![](ex9902img020.jpg)

Drugs with high SIs chosen for testing against Variants of Concern and small animal studies E7449 KYA1797K IWP-01 Yamaguchi et al, 2020

![](ex9902img021.jpg)

S A R S - C o V - 2 v i r u s t it e r (P F U / m l , L o g) U K B .1 .1 .7 6 5 (A lp h a) \* \* \* \* \* \* 4 \* 3 2 DMSO IW P - O 1 K YA 1797K S A R S -C o V -2 v ir u s tite r (P F U /m l, L o g) S A R S - C o V - 2 v ir u s t it e r (P F U /m l, L o g) Peroxisome-modulating drugs are effective against SARS-CoV-2 Variants of Concern P y r v in iu m In d ia B .1 .6 1 7 .2 (D e lt a) 6 \* \* 5 \* 4 \* \* 3 2 1 \* 0 DMSO IW P - O 1 K YA 1797K \* P y r v in iu m B .1 .1 .5 2 9 (O m ic ro n) 5 \* 4 \* \* \* \* \* 3 \* 2 1 \* 0 DMSO IW P -O 1 K YA 1797K P y r v in iu m Xu, Elaish, Wong et al, in preparation

![](ex9902img022.jpg)

-0 8 4 6 KYA1 797K ETC1922 159 P y rv in iu m iC R T -1 4 SM 04 690 E744 9 NCB 974 W n tC59 LGK- 0 DMS O IW P O1 3 \* 9 3 E744 974 W n tC59 NCB -0 8 4 6 KYA1 797K ETC1922 159 P y rv in iu m iC R T -1 4 SM 04 690 LGK- 9 DMS O IW P O1 E744 0 9 NCB -0 8 4 6 KYA1 797K ETC1922 159 P y rv in iu m iC R T -1 4 SM 04 690 O IW P O1 LGK974 W n tC59 DMS V ir u s t it e r (P F U /m l, L o g) 2 E744 -0 8 4 6 KYA1 797K ETC1922 159 P y rv in iu m iC R T -1 4 SM 04 690 NCB 974 W n tC59 LGK- DMS O IW P O1 V ir u s t it e r (P F U /m l, L o g) Some Wnt/b-catenin inhibitors reduce replication of other human coronaviruses H C O V - N L 6 3 (C a lu 3) 10 µm 5 1 µm 4 \* \* 1 \* H C O V - 2 2 9 E (C a lu 3) 10 µm 5 1 µm 4 \* 2 1 \*

![](ex9902img023.jpg)

5 9 7 y E in rv 4 iu 4 m K 7 9 7 1 A P S D M 4 7 1 O 9 4 4 m in rv P y A Y K E 9 7 1 P IW iu 7 1 -O O S M K 5 \*\* -O \* \* \* 6 Y 6 \* P \* 7 K \* Alphavirus 8 IW \* M A Y V t itr e (P F U /m L , L o g) Flavivirus 7 D Z IK V titr e (P F U /m L , L o g) Wnt/b-catenin inhibitors reduce replication of other RNA viruses Xu, Elaish, Wong et al, in preparation

![](ex9902img024.jpg)

In vivo testing of Wnt inhibitors Drugs administered intranasally to female BALB/c mice (5 in each group) Intranasal challenge with 5 x 103 pfu of mouse-adapted SARS-CoV-2

![](ex9902img025.jpg)

KYA1797K § Destabilizes b-catenin by activating Axin-GSK3b complex § Tested via IP administration in mice (20 mg/kg/day) ú Here limited to 3 mg/kg due to solubility

![](ex9902img026.jpg)

E7449 § Other names: Stenoparib § Dual inhibitor of PARP1/2 & tankyrase1/2 § Orally bioavailable § Phase1/2 study for cancer indications ú Well tolerated (50-800 mg dosing) in humans ú 0% cytotoxicity in human cells at 10 µM in vitro

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Wnt inhibitors have modest effect on virusinduced weight loss N o r m a liz e d w e ig h t (%) 110 D M S O /S F M 100 D M S O /S A R S - C o V -2 E 7 4 4 9 /S A R S - C o V -2 90 6 mg/kg 80 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 T im e a f t e r c h a ll e n g e (d) N o r m a liz e d w e ig h t (%) 110 D M S O /S F M 100 D M S O /S A R S - C o V -2 K Y A 1 7 9 7 K /S A R S - C o V -2 90 3 mg/kg 80 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 T im e a f t e r c h a ll e n g e (d) Xu, Elaish, Wong et al, in preparation

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Anesthesia/IN drug administration causes mild transient weight loss N o r m a liz e d w e ig h t (%) 110 D M S O /S F M K Y A 1 7 9 7 K /S F M 105 E 7 4 4 9 /S F M 100 95 90 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 T im e a f t e r c h a ll e n g e (d)

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Wnt inhibitors reduce viral load in lungs PMP70 Nucleocapsid \*\* \*\*\* 6 5 4 Uninfected DMSO 9 44 E7 K YA 17 M 97 K SO 3 D SARS-CoV-2 titer (PFU/g tissue, Log) 4 DPI, Lung, N=5 Xu, Elaish, Wong et al, in preparation E7449 KYA1797K

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Wnt inhibitors reduce proinflammatory markers in lungs 40 Host Gene RNA (RFC) 8 30 20 10 0 6 4 2 SARS-CoV-2 K Y A 17 97 K E7 44 9 O D M S O S M D K Y A 17 97 K E 74 49 O S M D M S O 0 D Host Gene RNA (RFC) IL-1β IL-6 SARS-CoV-2 4-days post-infection Xu, Elaish, Wong et al, in preparation

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Wnt inhibitors reduce expression of miRNAs suppress peroxisome Wntthat inhibitors suppress expression biogenesis of PEX-targeting miRNAs (No infection)biogenesis HIV-1 Vpu suppresses peroxisome 4 3 2 Fujiki et al, 2014 1 \* \* \* \* \* \* \* \* \* \* D miR-500a-5p miR-34c-3p \* \* \* \* DM S m IWP O iR -O -9 1 Py K3Y-3 IW rvin Ap iu PO1 m +K YA DM m IW SO iR P -3 -O1 8 Py K1Y-3 IW rvin Ap iu PO1 m +K YA DM m IW SO iR P -4 -O1 8 Py K3Y-5 IW rvin Ap iu PO1 m +K YA iu PO1 m +K YA DM m IW SO iR P-3 O1 Py 4cK-Y 3 IW rvin Ap iu PO1 m +K YA 0 m I MSO iR W -5 P-O 00 1 Py aK-Y 5 IW rvin Ap Relative miRNA level (Fold) Flavivirus miR-93-3p Kristina Hopfensperger, and Daniel Sauter mBio 2020; doi:10.1128/mBio.00967-20 miR-381-3p miR-483-5p

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Summary § Inhibition of Wnt/b catenin pathway induces peroxisomes and enhances IFN response during viral infection § Significantly reduces SARS-CoV-2 replication in vitro and in vivo § Broad-spectrum activity against other RNA viruses

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Potential benefits of targeting peroxisomes for antiviral therapy § Drug candidates with good safety profiles ú Wnt inhibitors ú Peroxisome proliferator-activated receptor agonists § Reduce inflammation? § Prophylactic and early therapeutic use? § Do not induce IFN in absence of viral infection

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Inducing peroxisome biogenesis may have multiple health benefits Zalckvar and Schuldiner, 2022

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Ongoing/Future studies § In vivo efficacy of post-infection administration of Wnt/b catenin inhibitors § Increase bioavailability of drugs? Oral? Nebulizer? Derivatives? § Testing other Wnt/b catenin inhibitors and peroxisome proliferators alone and in combination § High throughput screening for novel peroxisome-inducing drugs

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Acknowledgements Dr. Zaikun Xu Dr. Mohamed Elaish Dr. Jason Wong Wil Branton Dr. Natacha Ogando Dr. David Evans Dr. Chris Power Ray Ishida Dr. Rick Rachubinski Dr. Barbara Knoblach Dr. Joaquin Lopez Orozco Dr. Lara Mahal Dr. Irv Mayers Dr. Ryan Noyce