WO2025210119A1

WO2025210119A1 - Pi3k and/or mtor inhibitors for treating viral disease

Info

Publication number: WO2025210119A1
Application number: PCT/EP2025/059059
Authority: WO
Inventors: David OLAGNIER; Ivan De Weber; Cedric BENY
Original assignee: Cortex Discovery GmbH; Aarhus Universitet
Current assignee: Cortex Discovery GmbH; Aarhus Universitet
Priority date: 2024-04-04
Filing date: 2025-04-03
Publication date: 2025-10-09
Anticipated expiration: 2026-10-04

Abstract

The present invention relates to panantiviral compounds, such as compounds providing a broad antiviral activity against a variety of viruses. More specifically the present invention relates to antiviral compounds directed against coronavirus and in particular SARS-type and MERS-type coronaviruses. More specifically, the present invention provides for the compounds PKI-179 and MTI-31 for use in a method of treatment, prevention or amelioration of a diseases or disorder caused by infection with a coronavirus such as SARS-CoV-2.

Description

PI3K AND/OR MTOR INHIBITORS FOR TREATING VIRAL DISEASE

Technical field

The present invention relates to pan-antiviral compounds, such as compounds providing a broad antiviral activity against a variety of viruses. More specifically the present invention relates to antiviral compounds directed against coronaviruses and in particular SARS-type and MERS-type coronaviruses. More specifically, the present invention provides for the compounds PKI-179 and MTI-31 for use in a method of treatment, prevention or amelioration of a diseases or disorder caused by infection with a coronavirus such as SARS-CoV-2.

Background

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a novel coronavirus that emerged in late 2019, was identified as the causative agent of the Coronavirus Disease 2019 (COVID-19). Initially reported in Wuhan, China, SARS-CoV- 2 swiftly spread worldwide, leading to the declaration of a pandemic by the World Health Organization (WHO) in March 2020, profoundly impacting public health, economies, and daily life worldwide. At the time of writing, there have been 774 million COVID-19 cases of which ~6.9 million have resulted in death.

Along with the time-consuming and costly nature of traditional drug development processes, this has prompted a significant emphasis on drug repurposing strategies, representing a more efficient approach for identifying potent SARS-CoV-2 inhibitors (Li et al., 2023).

One major challenge in drug repurposing is that most repurposed candidates lack proper validation (in physiologically relevant models), which aligns with the issue of many repurposed drugs failing to demonstrate clinical benefits during the COVID-19 pandemic, for example, seen for hydroxychloroquine and ivermectin (Dinesh Kumar et al., 2022; Reis et al., 2022; Self et al., 2020; Williams and Zhan, 2023). This reinforces the need for cautiousness and the requirement of robust in vitro and pre-clinical validation before considering the off-label use of existing drugs for new conditions.

Despite the development and deployment of different vaccine strategies against COVID-19, the search for specific antiviral drugs targeting the SARS-CoV-2 virus remains essential for treating COVID-19, addressing emerging variants, and providing therapeutic options for diverse populations (Li et al., 2023). Thus there remains a need in the field for development of effective pan-antiviral drugs targeting COVID-19 infections including emerging variants.

Summary

The present invention aims to solve the above identified problem by providing a number of repurposed drugs not previously recognized or known for any antiviral activity.

In a first aspect, the present invention provides a pharmaceutical composition consisting of: a. one or more small-molecule PI3K inhibitor and/or mTOR inhibitor; and b. optionally, one or more therapeutically inactive excipients, for use in a method of treatment, prevention and/or amelioration of a viral disease or infection, with the proviso that the small-molecule drug is not rapamycin.

In a second aspect, the present invention provides a pharmaceutical composition comprising a combination of PKI-179 and MTI-31 for use as a medicament.

In a third aspect, the present invention provides a pharmaceutical composition comprising a combination of PKI-179 and MTI-31 for use in a method of treatment, prevention and/or amelioration of a viral disease or infection.

Within the scope of the present invention, the viral disease or infection is preferably caused by a coronavirus. In particular the experimental framework presented in the examples herein support that the drugs designated as PKI-179 and MTI-31 provide a superior and surprising (pan-)antiviral effect against a variety of coronaviruses including SARS-CoV-2 which is superior to the known mTOR inhibitor rapamycin, and which could not have been expected a priori. In particular none of PKI-179 or MTI-31 were previously known to provide such profound antiviral effects in a variety of virus strains.

Thereby as provided herein, the one or more small-molecule drug is a PI3K inhibitor/blocker and/or mTOR inhibitor/blocker. In one embodiment, such a small- molecule drug may on account of its action against viruses such as SARS-CoV-2 be designated as an antiviral drug.

Description of Drawings

Figure 1 : In vitro antiviral evaluation of 6 potential repurposing compounds.

A-B: Correlation between drug concentration, cytotoxicity, and SARS-CoV-2 viral RNA expression for the 6 evaluated compounds tested on (A) VeroE6 hTMPRSS2 and (B) Calu3 cell lines. On each figure, relative viral RNA levels are represented in black and can be read-out on the left-hand axis, whereas LDH activity is represented in grey and can be read-out on the right-hand axis. Compounds were added 1 hour prior to SARS- CoV-2 infection at MOI 0.01 as described in the examples. Cytotoxicity was measured in the absence of SARS-CoV-2 infection by Lactate Dehydrogenase (LDH) assay after 24h. SARS-CoV-2 viral RNA expression was determined by qPCR 24 hours-post- infection (hpi) and compared to housekeeping gene TBP as a control. For every drug concentration tested, we included a control using an equivalent amount of DMSO under matching conditions.

C: Table displaying all IC50 for the different drugs tested on each cell line

Figure 2: MTI-31 and PKI-179 inhibit SARS-CoV-2 replication and outperforms the antiviral effect of rapamycin.

A: Calu3 cells were pre-treated for 1 h with 1 pM of MTI-31 or PKI-179 and subsequently infected with SARS-CoV-2 at MOI 0.01 for 24h. Viral replication was assessed by endpoint dilution assay in the supernatant of infected cells. Data are means ± SEM from three independent experiments (n=3).

B: Calu3 cells were seeded on glass coverslips and allowed to adhere overnight. Cells were treated with 1 pM of MTI-31 or PKI-179 for 1 h before SARS-CoV-2 infection at MOI 0.01. Cells treated with an equivalent amount of DMSO were used as a control. The SARS-CoV-2 nucleocapsid protein was visualized by confocal imaging at 48 hpi. Nuclei were stained using DAPI. Scalebar corresponds to 100 pm. Data are representative images of two independent experiments. Comparing with the DMSO control sample, the cells treated with PKI-179 or MTI-31 show a distinguishable reduction in SARS-CoV-2 nucleocapsid protein.

C-D: Calu3 cells were treated with MTI-31 , PKI-179 or rapamycin at 1 pM concentration for 1h prior to SARS-CoV-2 infection (MOI 0.01) for 24h. (C) SARS-CoV-2 viral RNA expression was determined by qPCR. Data are means ± SEM from two experiments performed in quadruplicate (n=2). (D) SARS-CoV-2 nucleocapsid protein expression and activation of the autophagy pathway were assessed by immunoblotting. Data are representative of three independent experiments. Statistics indicate significance by Student t-test for (A) and (C): ***P < 0.001.

Figure 3: MTI-31 and PKI-179 act prophylactically and therapeutically against SARS-CoV-2 in vitro.

A: Schematic representation of time of addition assay, illustrating treatment intervals and SARS-CoV-2 infection.

B-C: (B) Vero E6 hTMPRSS2 and (C) Calu3 cells were treated with 1 pM of MTI-31 , PKI- 179, or corresponding DMSO concentrations at the indicated time points and challenged with SARS-CoV-2 at a MOI of 0.01 for 24h. Cell lysates were assessed by immunoblotting for expression of SARS-CoV-2 nucleocapsid and autophagy markers. Data are representative of two independent experiments.

Figure 4: Antiviral effects of MTI-31 and PKI-179 on SARS-CoV-2 variants of concern.

Calu3 cells were treated with 1 pM of MTI-31 or PKI-179 for 1h before infection with variants of concern (MOI 0.01).

A: SARS-CoV-2 (Wuhan-like early European B.1 lineage (FR-4286))

B: Delta (B.1.617.2)

C: Omicron (B.1.1.529 BA.1)

Viral RNA expression was determined by qPCR. Data are means ± SEM from three independent experiments (n=3). Statistics indicate significance by Student t-test for (A) and (B): **P < 0.01.

Figure 5: MTI-31 and PKI-179 broadly suppress the replication of human pathogenic coronaviruses.

Vero-81 or Huh-7 cells were pretreated for 1h in presence of 1 pM of PKI-179 (A+C) or MTI-31 (B+D). Then Vero-81 cells were inoculated by SARS-CoV (MOI 0.05) and Huh- 7 cells by HCoV-229E (MOI 0.05) or MERS-CoV (MOI 0.05) for 24h.

A-B: Viral genomes quantification by qRT-PCR.

C-D: Infectious titers determined by TCID50. Data are means ± SEM of three independent experiments performed in biological duplicates. Statistical analysis of data in (A-D) was performed using a Kruskal-Wallis test: * p<0.05, **p<0.01 , ***p<0.001.

Figure 6: MTI-31 and PKI-179 reduce SARS-CoV-2 infection in primary human airway epithelial cultures derived from healthy donors

A: Graphical representation of the collection and culture of the nasal epithelial cells from healthy donors for the development of the primary human airway epithelial (HAE) culture model. The schematic was made using BioRender.com.

B: Schematic representation of compound administration and SARS-CoV-2 infection.

C: Primary HAE cultures derived from healthy donors were treated with 10 pM of MTI- 31 or PKI-179 for 3h before challenge with SARS-CoV-2 (MOI 0.1). SARS-CoV-2 viral RNA expression was determined at 24 hpi by qPCR. Data are means ± SEM of two independent experiments performed in technical duplicates (n=2). Statistics indicate significance by Student’s t-test: *p < 0.05. Experiment was performed on two different donors with a similar trend.

Detailed description

It has surprisingly been found through the experimental framework presented in the present disclosure, that small-molecule drugs inhibiting PI3K and/or mTOR have a pronounced antiviral effect against a broad spectrum of human coronavirus and in particular new coronavirus SARS-CoV-2. The present inventors have performed comparative testing with other antimicrobial drugs either in Vero E6 cells expressing human TMPRSS2 or in human lung epithelial cell line Calu-3 and found that the compounds classified as PI3K and/or mTOR inhibitors possess a superior potent antiviral effect.

Furthermore, within the group of mTOR inhibitors, the compounds designated as MTI- 31 and PKI-179 also demonstrated an antiviral effect superior to other well-known drugs such as rapamycin which could not have been expected or predicted a priori.

Small molecule drugs

It is within the scope of the present invention to provide pharmaceutical compositions comprising or consisting of therapeutically active small-molecule drugs and/or compounds. As used herein, the term “comprising” is also to be understood as being a disclosure to the same embodiment in a limited “consisting of’ manner.

One embodiment of the present disclosure is provided by a pharmaceutical composition consisting of: a. one or more small-molecule PI3K inhibitor and/or mTOR inhibitor; and b. optionally, one or more therapeutically inactive excipients; for use in a method of treatment, prevention and/or amelioration of a viral disease or infection, with the proviso that the small-molecule drug is not rapamycin.

As used herein, the term “therapeutically inactive” regarding excipients and/or absorption promotors are intended to underline that the therapeutic effect observed resides in the small-molecule PI3K inhibitor and/or mTOR inhibitor. That is to say if the small-molecule PI3K and/or mTOR inhibitor is absent from the pharmaceutical composition, no antiviral therapeutic effect can be observed originating from the therapeutically inactive excipients and/or absorption promotors.

In one embodiment, the one or more small-molecule inhibitor is an mTOR inhibitor and/or blocker. In one embodiment, the one or more small-molecule inhibitor is a PI3K inhibitor and/or blocker. In one embodiment, the one or more small-molecule inhibitor is a dual PI3K/mTOR inhibitor and/or blocker. The purpose of the PI3K and/or mTOR inhibitor functionality is to provide an antiviral effect.

In one embodiment of the present disclosure, the therapeutic drug is selected from the group consisting of

PKI-179 MTI-31

Or a pharmaceutically acceptable prodrug, ester, salt or hydrate thereof.

In one embodiment of the present disclosure, the therapeutic drug is PKI-179 or a pharmaceutically acceptable prodrug, ester, salt or hydrate thereof.

PKI-179

PKI-179 may also be characterized by the systematic name 1-(4-(4-(3-oxa-8- azabicyclo[3.2.1]octan-8-yl)-6-morpholino-1 ,3,5-triazin-2-yl)phenyl)-3-(pyridin-4-yl)urea, or by CAS No.: 1197160-28-3 .

In one embodiment of the present disclosure, the therapeutic drug is MTI-31 or a pharmaceutically acceptable prodrug, ester, salt or hydrate thereof.

MTI-31

MTI-31 may also be characterized by the systematic name 3-(4-(3-oxa-8- azabicyclo[3.2.1]octan-8-yl)-2-((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-N- methylbenzamide, or by CAS No.: 1567915-38-1.

One embodiment of the present disclosure is provided by a pharmaceutical composition consisting of: a. PKI-179 or a pharmaceutically acceptable prodrug, ester, salt or hydrate thereof; and b. optionally, one or more therapeutically inactive excipients; for use in a method of treatment, prevention and/or amelioration of a viral disease or infection, preferably wherein the viral disease or infection is caused by a coronavirus.

One embodiment of the present disclosure is provided by a pharmaceutical composition consisting of: a. MTI-31 or a pharmaceutically acceptable prodrug, ester, salt or hydrate thereof; and b. optionally, one or more therapeutically inactive excipients; for use in a method of treatment, prevention and/or amelioration of a viral disease or infection, preferably wherein the viral disease or infection is caused by a coronavirus.

An embodiment of the present disclosure also provides use of any one of PKI-179 or MTI-31 (alone or in combination) as an antiviral agent.

In one embodiment of the present disclosure, the small-molecule drug is characterized by an IC50 of 50 pm or less, such as 40 pm or less, such as 30 pm or less, such as 25 pm or less, such as 20 pm or less, such as 10 pm or less, such as 5 pm or less, such as 1 m or less, such as 0.5 pm or less, in particular wherein the IC50 is evaluated against SARS-CoV-2.

In one embodiment of the present disclosure, the small-molecule drug is characterized by an IC50 of 1 pm or less, preferably 0.5 pm or less, in particular wherein the IC50 is evaluated against SARS-CoV-2.

In one embodiment of the present disclosure, the pharmaceutical composition does not comprise a compound of any one of formula (I) and/or formula (II) as defined in WO2022/219157 which is incorporated herein by reference.

Importantly, the compounds MTI-31 and PKI-179 were found to exhibit minimal toxicity, except for a notable observation with PKI-179 at higher concentrations in VeroE6 hTMPRSS2. Notably, MTI-31 and PKI-179 has emerged within the framework of the experimental section presented in the present disclosure as potent antiviral candidates, displaying remarkable antiviral efficacy even at low concentrations tested in Vero E6 cells expressing human TMPRSS2 and in human lung epithelial cell line Calu-3.

Further exploring the antiviral activity of PKI-179 and MTI-31 , time-of-addition assays revealed that these compounds inhibit SARS-CoV-2 infection at a post-entry step in the virus replication cycle, emphasizing their sustained antiviral impact.

Furthermore, PKI-179 and MTI-31 showed considerable inhibitory potential against also Delta and Omicron SARS-CoV-2 variants of concern. The pan-antiviral action of these compounds was further confirmed against various previously known human coronaviruses, including SARS-CoV, MERS-CoV, and HCoV-229E, underscoring their broad-spectrum antiviral capabilities.

The present disclosure has tested and verified PKI-179 and MTI-31 , two PI3K and/or TORC1/2 (mammalian target of rapamycin) inhibitors, as potent antiviral drugs acting broadly on coronaviruses.

Interestingly, PKI-179 and MTI-31 demonstrated superior efficacy in inhibiting SARS- CoV-2 infection compared to rapamycin, a well-established mTOR inhibitor. The heightened effectiveness at low micromolar concentration suggested their potential as antiviral agents. Differences in their inhibitory mechanisms within the mTOR pathway and potential off-target effects may contribute to the varying levels of antiviral activity observed. These molecular insights highlighted PKI-179 and MTI-31 as promising candidates for further investigation, with their comparison to rapamycin emphasizing their potential for expedited clinical evaluation.

These surprising results demonstrates a highly effective reduction of coronavirus, at much lower concentrations than observed for e.g., rapamycin thereby potentially minimizing the drug concentrations required to obtain the desired antiviral effect and associated risk of drug-related adverse effects. For instance rapamycin is known to be associated with several adverse effects, ranging from minor ailments to very severe pathologies including suspected carcinogenic and neurodegenerative effects.

Without wishing to be bound by theory, it is also considered as made credibly plausible that a combination treatment combining PKI-179 and MTI-31 would have a superior antiviral effect against a broad range of coronaviruses. The present disclosure thereby also provides an embodiment in the form of a pharmaceutical composition comprising a combination of PKI-179 and MTI-31 for use as a medicament. Further is provided in an embodiment, a pharmaceutical composition comprising a combination of PKI-179 and MTI-31 for use in a method of treatment, prevention and/or amelioration of a viral disease or infection, preferably wherein said disease or infection is caused by coronavirus, such as by SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E or a subvariant of any of the foregoing.

Treatment

It is within the scope of the present invention to provide a composition for use in a method of treatment of a disease or infection caused by a virus. The inventors have shown that certain small-molecule drugs serving the function of inhibiting PI3K and/or mTOR signalling pathways provide an unexpected superior therapeutic effect against certain type of viruses compared to state-of-the-art rapamycin.

Also within the scope of the present invention are method of treatment, prevention and/or amelioration of a viral disease or infection in a subject in need thereof, optionally wherein the virus is a coronavirus, the method comprising administering to the subject a therapeutically effective amount of a small-molecule drug which is a PI3K inhibitor and/or mTOR inhibitor. Also within the scope of the present invention are use of a composition comprising or consisting of a therapeutically effective amount of a small-molecule PI3K inhibitor and/or mTOR inhibitor for the manufacture of a medicament for the treatment, prevention and/or amelioration of a viral disease or infection, optionally wherein the virus is a coronavirus.

In one embodiment the method of treatment as described herein may involve blocking and/or inhibition of PI3K and/or mTOR signalling pathways including any downstream pathways.

Examples of disease-causing virus include coronavirus, adenovirus, picornavirus, flavivirus, herpesvirus, filovirus, poxvirus and retrovirus. Preferably the compositions of the present disclosure are for use in a method of treatment of viral diseases or infections caused by coronavirus.

In one embodiment, the compositions disclosed herein are for use in the treatment of a coronavirus of the type MERS-CoV, SARS-CoV, SARS-CoV-2, HCoV-229E, HCoV- NL63, HCoV-0043 and HCoV-HKU1.

In one embodiment, the compositions disclosed herein are for use in the treatment of a coronavirus of the type SARS-CoV-2. Infections caused by SARS-CoV-2 may commonly be referred to as COVID-19 infections and could be classified by the SARS- CoV-2 lineage and/or variant, such as wherein the variant of SARS-CoV-2 may be selected from wild-type, alpha, beta, delta or omicron.

In one embodiment the variant is omicron. In one embodiment, the omicron variant is a virus of B.1.1.529 lineage, such as B.1.1.529 or B.1.1.529.1 (BA.1).

In one embodiment the variant is delta. In one embodiment, the delta variant is a virus of B.1.617.2 lineage.

In one embodiment the variant is beta. In one embodiment, the beta variant is a virus of B.1.351.1 lineage. Subject

The pharmaceutical composition of the present disclosure is for use in a method of treatment of viral diseases or disorders as described herein in a subject in need thereof.

In one embodiment, the subject is infected with a coronavirus. In another embodiment, the treatment is prophylactic and/or preventive and is administered to a subject in risk of being infected with a viral infection such as a coronavirus, more preferably SARS- CoV-2.

In one embodiment, the subject is infected with a coronavirus such as SARS-CoV-2 and/or MERS-CoV and/or HCoV-229E.

In one embodiment the subject is a mammal. In particular the subject may be a human.

In one embodiment, the subject intended to be treated and/or to be administered the composition for the treatment is elderly, such as is above 50 years of age, such as above 60 years of age, such as above 65 years of age, such as 70 years of age or above. In one embodiment, the subject may suffer from an immunodeficiency and/or be immunosuppressed.

Routes of administration

It will be appreciated that the preferred route of administration will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated, the location of the tissue to be treated in the body and the active ingredient chosen.

In one embodiment, the administration is by enteral administration or parenteral administration, and the pharmaceutical formulation of the present disclosure may be formulated for such administration. Appropriate dosage forms for such administration may be prepared by conventional techniques.

In one embodiment of the present disclosure, the composition is formulated for parenteral administration, such as intravenous, subcutaneous or intramuscular administration. In one embodiment of the present disclosure, the composition is formulated for enteral administration such as oral, rectal or nasogastric administration.

In one embodiment of the present disclosure, the composition is formulated for topical administration such as pulmonary, intranasal, or intratracheal administration.

Oral administration

Oral administration is normally for enteral drug delivery, wherein the compound is delivered through the enteral mucosa. Syrups and solid oral dosage forms, such as tablets, capsules, and the like, are commonly used.

In one embodiment, the pharmaceutical composition comprising or consisting of any one of PKI-179 and/or MTI-31 is formulated and/or administered by oral administration.

Parenteral administration

Parenteral administration is any administration route not being the oral/enteral route whereby the medicament avoids first-pass degradation in the liver. Accordingly, parenteral administration includes any injections and infusions, for example bolus injection or continuous infusion, such as intravenous administration, intramuscular administration, subcutaneous administration. Furthermore, parenteral administration includes inhalations and topical administration.

In one embodiment of the present disclosure, the pharmaceutical composition may be administered topically to cross any mucosal membrane of an animal to which the biologically active substance is to be given, e.g. in the mouth or lungs.

In one embodiment, the pharmaceutical composition comprising or consisting of any one of PKI-179 and/or MTI-31 is administered by parenteral administration.

In one embodiment, the pharmaceutical composition comprising or consisting of any one of PKI-179 and/or MTI-31 is administered by topical administration. Dosage

According to the present disclosure, the composition of the invention comprising the small-molecule inhibitor drugs is administered to individuals in need of treatment in pharmaceutically effective doses. A therapeutically effective amount of a compound is an amount sufficient to cure, prevent, reduce the risk of, alleviate or partially arrest the clinical manifestations of a given viral disease or infection and its complications. The amount that is effective for a particular therapeutic purpose will depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. The compound may be administered one or several times per day, such as from 1 to 8 times per day, such as from 1 to 6 times per day, such as from 1 to 5 times per day, such as from 1 to 4 times per day, such as from 1 to 3 times per day, such as from 1 to 2 times per day, such as from 2 to 4 times per day, such as from 2 to 3 times per day. Alternatively, the compound may be administered less than once a day, for example once a day, such as once every second day, for example once every third day, such as once every fourth day, for example once every fifth day, such as once every sixth day, for example once every week.

In one embodiment the composition comprising the small-molecule drugs of the invention is administered in a therapeutically effective amount, such as in an amount of 0.1 pg to 100 mg of a small-molecule.

It follows that in one embodiment the composition comprising the small-molecule drugs of the invention is administered in an amount of 0.1 pg to 100 mg, such as 0.1 pg to 1 pg, such as 1 pg to 10 pg, such as 10 pg to 20 pg, such as 20 pg to 50 pg, such as 50 pg to 100 pg, such as 100 pg to 200 pg, such as 200 pg to 500 pg, such as 500 pg to 1 mg, such as 1 mg to 5 mg, such as 5 mg to 10 mg, such as 10 mg to 50 mg, such as 50 mg to 100 mg per day.

Per day means the dosage may be given in one dosage or divided in multiple dosages per day, including once a day (QD), twice a day (BID) and/or three times a day (TID).

In another embodiment the composition comprising the small-molecule drugs of the invention is administered in a dosage of from 1 pg/kg -10,000 pg/kg body weight, such as 1 pg/kg - 7,500 pg/kg, such as 1 pg/kg - 5,000 pg/kg, such as 1 pg/kg - 2,000 pg/kg, such as 1 pg/kg - 1 ,000 pg/kg, such as 1 pg/kg - 700 pg/kg, such as 5 pg/kg - 500 pg/kg, such as 10 pg/kg to 100 pg/kg bodyweight.

Pharmaceutical composition

Whilst it is possible for the small-molecule drugs of the present invention to be administered as the free-base drug or a pharmaceutically acceptable prodrug, ester, salt or hydrate thereof, it is preferred to present them in the form of a pharmaceutical formulation comprising one or more therapeutically inactive excipients and/or absorption promotors. Accordingly, the present invention further provides a pharmaceutical formulation, consisting of at least one or more small-molecule PI3K inhibitor and/or mTOR inhibitor or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable therapeutically inactive excipients. The pharmaceutical formulations may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, (ISBN: 978-0-12- 820007-0).

As used herein, the term “excipient” in the context of therapeutically inactive excipient, is to be understood as referring to ingredients with no medical/therapeutic effect on the disease or infection to be treated, but solely used in drug formulations to improve e.g., drug stability, bioavailability, absorption, taste, appearance, and ease of administration. As such, an excipient is a substance formulated alongside the active ingredient of a medicament, included exemplary for the purpose of bulking up formulations that contain potent active ingredients in relatively small amounts (thus often referred to as "bulking agents", "fillers", or "diluents"), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as but not limited to facilitating drug absorption, reducing viscosity, or enhancing solubility.

In the case of solid oral dosage forms such as tablets, granules or capsules, the excipient may be provided for the purpose of acting as one or more of binders, fillers, diluents, disintegrant, lubricant, film former, coating, plasticizer and wetting agent.

As used herein, a “binder” is an excipient that acts as an adhesive agent to bind together the powdered ingredients, most often used in a tablet or granule formulation. Binders impact the mechanical integrity of solid dosage forms by imparting cohesiveness to the powdered mixture. Additionally, binders may contribute to controlling the rate of drug release by influencing the porosity and permeability of the tablet matrix.

As used herein, a “filler” and/or “diluent” is an inactive ingredient added to a pharmaceutical formulation to increase the bulk volume of the dosage form without significantly affecting its therapeutic activity. It will be readily clear to a person of skill in the art that some excipients are not exclusively binder or filler but may have physical properties belonging to either class. It is therefore suitable to group some of these excipients together in appropriate terms.

In one embodiment of the present disclosure, the excipient in the form of a binder and/or filler and/or diluent may comprise one or more selected from lactose, starch, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), dextrose, fructose, hypromellose, maltodextrin, sucrose, sorbitol and xylitol, dicalcium phosphate, hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), microcrystalline cellulose, mannitol, maltose, maltitol, isomalt, and gelatin.

As used herein, a “disintegrant” is known in the art to expand and dissolve when wet causing the compressed pharmaceutical composition, such as a tablet, to break apart.

In one embodiment of the present disclosure, the excipient in the form of a disintegrant may comprise one or more selected from croscarmellose, croscarmellose sodium, sodium starch glycolate, povidone, and crospovidone.

As used herein, a “lubricant” is an excipient that prevents ingredients from sticking to and clogging the tablet punches or capsule filling machine. During tablet production, lubricants also ensure that tablet formation and ejection can occur with low friction between the solid and die wall.

In one embodiment of the present disclosure, the excipient in the form of a lubricant may comprise one or more selected from magnesium stearate, silicon dioxide, sodium lauryl sulfate and talc. As used herein, an “absorption promoter” is an excipient used in oral drug formulations to improve drug absorption particularly for drugs with low solubility or permeability, thereby ultimately enhancing therapeutic efficacy.

In one embodiment of the present disclosure, the excipient in the form of an absorption promotor may comprise one or more selected from chitosan, hydroxypropyl cellulose (HPC), sodium lauryl sulphate, polysorbate 80, sodium caprylate, sodium taurocholate, cyclodextrins such as hydroxypropyl beta-cyclodextrin, and phospholipids, such as phosphatidylcholine.

In the case of liquid dosage forms such as i.v. injections the excipient may be provided for the purpose of acting as one or more of carriers, electrolytes, and pH adjusters.

In one embodiment of the present disclosure, the excipient in the form of a carrier may comprise one or more of water, ethanol, glycerol and polyethylene glycol. In one embodiment of the present disclosure, the excipient in the form of an electrolyte and/or pH adjuster may comprise one or more of sodium chloride, potassium chloride, calcium chloride, sodium bicarbonate, sodium acetate, magnesium sulphate, sodium citrate, and glucose.

Formulation

The composition comprising the small-molecule drugs of the invention may be administered in any suitable way e.g., orally, sublingually, or parenterally, and it may be presented in any suitable form for such administration, e.g., in the form of solutions, suspension, tablets, capsules, powders, syrups or dispersions for injection such as intravenous injection.

In one embodiment, the composition comprising the small-molecule drugs of the invention is formulated as a suspension.

In one embodiment, the composition comprising the small-molecule drugs of the invention is formulated as an oral dose form, such as a solid oral dose form or pharmaceutical entity, such as tablets, granules or capsules, or a liquid oral dose form. Methods for the preparation of solid pharmaceutical preparations are well known in the art. In another embodiment the composition comprising the small-molecule drugs of the invention is formulated as a liquid injectable dose form.

In one embodiment, the pharmaceutical composition of the invention consists of the at least one small-molecule PI3K and/or mTOR inhibitor drug and one or more therapeutically inactive excipients independently selected from binders, fillers, diluents, disintegrants, lubricants, film formers, coatings, plasticizers, absorption promoters, wetting agents, carriers, electrolytes, and pH adjusters.

Examples

Material and Methods

Selected Compounds

Oteseconazole (HY-17643), LHF-535 (HY-112762), PKI-179 (HY-11080) MTI-31 (HY- 126077), etalocib (HY-13628), and fosnetupitant (HY-17615) were purchased from MedChem Express. Compounds were dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 10 mM and stored at -80°C. Matching DMSO conditions were used as the appropriate control conditions in all experiments.

Cell culture

Vero E6 cells expressing human TMPRSS2 (VeroE6 hTMPRSS2) were kindly provided by Makoto Takeda (Matsuyama et al., 2020) and Calu3 epithelial lung cancer cells were kindly provided by Laureano de le Vega, Dundee University, Scotland, UK. Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (ThermoFisher) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich), 1000 U ml-1 penicillin (Gibco), 1000 pg ml^-1 streptomycin (Gibco), and 2 mM L-glutamine (Gibco). For VeroE6 hTMPRSS2 cells, cell culture media was supplemented with 10 ug/mL blasticidin (to maintain TMPRSS2 expression) (Invivogen). Cells were maintained at 37° with 5% CO2. Huh-7 cells, a hepatocellular carcinoma cell line and Vero-81 cells, an African green monkey cell line (ATCC CCL-81), were grown in DMEM (Life Technologies) supplemented with 10% fetal bovine serum and 2 mM glutamax (Life Technologies) at 37°C with 5% CO2. HAE-ALI culture

The Human Airway Epithelium (HAE)-Air-Liquid interface (ALI) model was generated and cultured as previously described (Carter-Timofte et al., 2021 ; Olagnier et al., 2020). Upon treatment, the apical side of fully differentiated HAE-ALI cultures was washed for 5 min using DMEM (low glucose, no additives) (ThermoFisher) to remove mucus. The basolateral medium was changed with fresh medium containing 10 pM of indicated drugs or DMSO and 100 pL DMEM containing 10 pM of indicated drugs or DMSO was added to the apical compartment. The pre-treatment was left for 3h. before also adding SARS-CoV2 at a multiplicity of infection (MOI) of 0.1 to the apical compartment. After 1 hour of infection, both the drug- and virus-containing solution were removed from the apical side, but the drugs were maintained in the basolateral chamber. After 24h, the cells were harvested using Trypsin/EDTA, 400 pl was added basolaterally and 150 pl was added apically, and the harvest was repeated apically until all cells were removed from the membranes. Cells were lysed for RNA isolation using lysis buffer and isolation kit as mentioned below. The data are representative of one experiment that was performed on two different donors.

SARS-CoV-2 and variants of concern

SARS-CoV-2 Wuhan-like early European B.1 lineage (FR-4286) was kindly provided by Professor Georg Kochs (University of Freiburg). Delta variant B.1.617.2 (SARS- CoV2/hu/DK/SSI-H11) was provided by Statens Serum Institut, SSI, Denmark. Omicron variant B.1.1.529 BA.1 (Omicron #01 , 0066-P2) was kindly provided by Prof. Alex Sigal, Africa Health Research Institute, AHRI, South Africa. The viruses were propagated as previously described (Carter-Timofte et al., 2021). Briefly, 10 x 10⁶ VerohTMPRSS2 cells were seeded in T175 cell culture flasks and infected the following day at a MOI of 0.005 in 10 mL of cell culture media containing serum. After 1 hour the cell culture medium was increased to 20 mL per flask and virus propagation continued for 72 hours. Cell debris was removed by centrifugation of the cell culture supernatants from the different flasks at 300 g for 5 min. Viruses were concentrated in Amicon filter tubes by spinning at 4000 g for 30 min at 4°C. The concentrated virus was further aliquoted and stored at -80°C. The amount of infectious virus was determined using an endpoint dilution assay (TCID50) as described below.

SARS-CoV, MERS-CoV and HCoV-229E The MERS-CoV (EMC12 strain) was generated by transfection of Huh-7 cells with a plasmid containing MERS-CoV full genome (kindly provided by Dr L. Enjuanes, National Center of Biotechnology, Spain). Larger viral stocks were produced in Huh-7 cells by inoculation at an MOI of 0.01. SARS-CoV (Frankfurt-1 strain, kindly provided by Dr M. Vialette, Institut Pasteur de Lille, France) was propagated in Vero E6 cells expressing TMPRSS2 by inoculation at a MOI of 0.01. HCoV-229E (VR-740 strain, ATCC) was propagated in Huh-7 cells by inoculation at a MOI of 0.01 and at 33°C. After lysis of the cells, cell supernatants were collected, centrifuged to remove cellular debris and aliquoted. MERS-CoV, SARS-CoV and HCoV-229E viral stocks were then stored at -80°C.

SARS-CoV-2 infection experiments

For infection experiments, the cells were seeded in a 24-well plate (respectively for VeroE6 hTMPRSS2 and Calu3) and were allowed to adhere overnight. Plating density: 100.000 Vero cells or 300.000 Calu3 cells per well in 500 uL of cell culture media (see above)

All tested compounds were dissolved into a 10 mM stock in DMSO. The respective compounds were subjected to a 1/100 pre-dilution in media to get a 100 uM stock and from there we added to the wells 1h prior to viral infection at indicated concentrations. Cells were infected with SARS-CoV-2 in a 10 uL volume in presence of the respective compound (MOI of 0.01). Tested compounds were MTI-31, Oteseconazole, PKI-179, Fosnetupitant, LHF-535 and Etalocib.

After 1h the medium was replaced with fresh DMEM containing the compounds. Readout was evaluated using LDH assay from supernatant and cells lysates for qPCR.

Endpoint dilution assay (TCID50) for SARS-CoV-2

An endpoint dilution assay (TCID50) was performed to determine the amount of infectious virus in cell culture supernatants or generated virus stocks. 2 x 10⁴ Vero hTMPRSS2 cells were seeded in 90 uL of DMEM in a 96 well plate. The following day, the cell culture media was replaced with DMEM containing 2% FCS. Samples were diluted 10x using 2% FCS DMEM and 10 uL of each dilution was added to the cells. Samples were further diluted by 10-fold serial dilutions in the 96-well plate to a final dilution range of 10'²-1 O'¹² in octuplicates. After 72 hours, the cytopathic effect (CPE) was evaluated using standard microscopy and the 50% tissue culture infectious dose was calculated using the Reed and Muench method as described in (Carter-Timofte et al., 2021). qPCR for SARS-CoV-2

Cells were washed with PBS and lysed in 300 pL of RNA lysis buffer (Roche) diluted with 200 pL of PBS, followed by RNA extraction and gene expression analysis. RNA was extracted using the High Pure RNA Isolation Kit (Roche) according to the manufacturer’s instructions. RNA samples were diluted 1/25 and gene expression was analyzed by real-time quantitative PCR using TaqMan® RNA-to-CT ™ 1-Step Kit (Applied Biosystems) as previously described (Carter-Timofte et al., 2021). For SARS- CoV-2 gene detection, primers and probe sequences were provided by the CDC and purchased from Eurofins. Samples were analyzed in a final volume of 10 pL, containing 5 pL of master mix, 0.5 pL of forward primer (10 pmol/pL), 0.7 pL of reverse primer (10 pmol/pL), 0.2 pL of probe (20 pmol/pL), 2.4 pL of nuclease-free water, and 1 pL of diluted RNA. The forward primer, reverse primer and probe employed for SARS-CoV-2 analyses are published in the Methods section of D. Olagnier et al., (2020). The analysis was performed on a ThermoFisher Scientific qPCR machine (Quant Studio 5). TATA-Box Binding Protein (TBP) was used as the invariant control and was obtained from ThermoFisher.

MERS-CoV, SARS-CoV, HCoV-229E infection experiments and TCID50 MERS-CoV (EMC12 strain) and HCoV-229E (VR740 strain, ATCC) infection assays were performed in Huh-7 cells and SARS-CoV infection in Vero-81 cells seeded in 24- well plates. Cells were pre-treated for 1h with indicated concentrations of compounds or DMSO. Then, cells were infected at a MOI of 0.05 for 1h in the presence of the compounds and the medium was replaced with fresh medium containing the compound. Twenty-four hours later, cell supernatants were harvested, and total cellular RNA was extracted by using the NucleoSpin RNA plus kit (Macherey Nagel) according to the manufacturer’s instructions. Infectious viral particles in the supernatant were titrated using the TCID50 method. qPCR for MERS-CoV, SARS-CoV and HCoV-229E

For MERS-CoV and HCoV-229E intracellular genome quantification, reverse transcription was performed using the high-capacity cDNA reverse transcription kit (Life Technologies) according to the manufacturer’s instructions. Then cDNA was subjected to qRT-PCR using the Taqman universal PCR mastermix (Life Technologies).

The forward primer, reverse primer and probe targeting the N gene and employed for MERS-CoV gene quantification are published in the Methods section of V. S. Raj et al., (2013).

The forward primer, reverse primer and probe employed for HCoV-229E M fragment amplification are published in the Materials and Methods section of L. Vijgen et al., (2005).

For SARS-CoV, genomes were quantified by using the Low Rox One-step RT-probe mastermix (Eurogenetec) with forward primers, revers primers and probe targeting E gene, published in Table 1 of V. M. Corman et al., (2020).

Immunoblotting

Immunoblotting was performed as previously described in (Olagnier et al., 2020). Briefly, cells were washed with PBS and subsequently lysed in 100 uL ice-cold Pierce RIPA lysis buffer (Thermofisher Scientific), supplemented with 10 mM NaF, 1x complete protease inhibitor cocktail (Roche) and 5 IIJ/mL benzonase (Sigma). Protein concentration was measured using a BCA Protein Assay Kit (Thermofisher Scientific). Whole-cell lysates were resuspended in a loading buffer, consisting of XT Sample Buffer (BioRad) and XT Reducing Agent (BioRad). Samples were then denatured at 95°C for 5 minutes. 10-40 pg of the reduced sample was separated by SDS-PAGE on a 4-20% CriterionTM TGX pre-casted gradient gels (BioRad). Each gel was first run at 70V for 20 min, following 110V for 50 min. Transfer onto a Midi Format 0,2 pM PVDF membrane (BioRad) was done using a Transblot Turbo Transfer System (BioRad) for 7 min. Membranes were blocked for 1h at room temperature with 5% skimmed milk (Sigma-Aldrich) in PBS supplemented with 0.05% Tween-20 (PBS-T). Membranes were fractionated into smaller pieces and incubated overnight at 4°C with one of the following primary antibodies in PBS-T and 0.02% sodium azide: anti-SQSTM1/p62 (#8025, Cell Signaling 1:1000), anti-LC3B (#3868, Cell Signaling 1 :1000), anti-SARS- CoV-2 Nucleocapsid (#26369, Cell Signaling, 1:1000) and anti-Vinculin (#V9264, Sigma 1:10.000) was used as a loading control. Membranes were washed 3 times in PBST for 15 min following incubation with secondary antibodies: HRP conjugated F(ab)2 donkey antirabbit IgG (H+L) or HRP conjugated F(ab)2 donkey anti-mouse IgG (H+L) (1:10.000) (Jackson ImmunoResearch) in PBS-T 1% skimmed-milk for 1h at RT. Membranes were washed 3 times in PBS-T for 10 min and subsequently incubated with SuperSignal West Dura Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) for 1 min prior to exposure using i Bright CL1500 Imaging System (ThermoFisher Scientific).

Confocal microscopy

Calu3 cells were seeded onto glass coverslips placed in the bottom of 24-well plates and pre-treated with either DMSO or 1 uM of MTI-31 or PKI-179 1h prior to SARS-CoV- 2 infection at MOI 0.01 as described above. After 48h, cells were washed with PBS and fixed for 30 min with 4% PFA at RT, following permeabilization with 0.2% Triton X-100 in PBS for 20 min. Subsequently, blocking with 2% goat serum (Sigma-Aldrich) in PBS was performed for 40 min. Following, cells were incubated with rabbit anti-SARS-CoV- 2 nucleocapsid (#26369, Cell Signaling 1:300) in the blocking solution for 1h at RT. Cells were washed 3 times with PBS and incubated with goat anti-rabbit Alexa Fluor 488 nm fluorophore-conjugated secondary antibody (#A11008, Invitrogen, 1:300) and PureBlu DAPI nuclear staining dye (#1351303, BioRad 1:100) for 1h at RT protected from light. Cells were washed 3 times with PBS and mounted onto microscope slides using Prolong Gold Antifade Mounting medium (Invitrogen). Slides were air-dried protected from light and examined using Zeiss LSM 710 inverted confocal microscope with corresponding Zeiss ZEN software.

LDH assay

Cellular toxicity of the respective compounds was measured 48h post-treatment using CyQUANT™ LDH Cytotoxicity Assay Kit (ThermoFisher) according to the manufacturer’s instructions. Untreated cells were used as a negative control, whereas cells lysed with the provided lysis buffer served as a positive control. LDH activity was determined following subtraction of the background 690 nm absorbance value from the 450 nm absorbance value measured on a BioTek Microplate Reader (BioTek Instruments). The percentage of cytotoxicity was calculated using the following formula: (LDH activity - LDH activity control) I (LDH activity positive control - LDH activity negative control) x 100.

Statistics and reproducibility

The data are shown as means of biological replicates ± standard error means (SEM).

The number of replicates is indicated within figure legends. Statistical significance between groups was determined using a two-tailed Student’s t-test for SARS-CoV-2 experiments. For experiments with MERS-CoV, SARS-CoV and HCoV-229E, statistical significance between the different treatment groups was assessed using Dunn’s test. All statistical analyses were performed using GraphPad Prism 9. ****P <0.0001, ***P <0.001 , **P < 0.01 , *P < 0.05; ns, not significant.

Example 1 - PKI-179 and MTI-31 suppress SARS-CoV-2 replication and outperforms the antiviral effect of rapamycin.

Following initial testing on VeroE6 hTMPRSS2 and Calu-3 cells (see above, SA S- CoV-2 infection experiments and Figs. 1A-1C)), specific focus was put on the two bestperforming drugs, PKI-179 and MTI-31.

To assess if PKI-179 and MTI-31 are able to inhibit the release of infectious virus at nanomolar (nM) concentrations, supernatants from Calu-3 cells infected with SARS- CoV-2 were collected following infection and drug treatment. An impressive ~40-fold reduction in the production of viral progeny was observed in PKI-179 and MTI-31 - treated cells, as determined by TCID50 assay (Fig. 2A).

Furthermore, immunofluorescence staining of viral nucleocapsid protein showed an intracellular absence of the protein in Calu-3 cells treated with either PKI-179 and MTI- 31 compared to untreated cells (Fig. 2B). Another mTORCI inhibitor, rapamycin, has previously been demonstrated to display some antiviral action against SARS-CoV-2 (Mullen et al., 2021). The in vitro antiviral activity of all three mTORC inhibitors, rapamycin, MTI-31 and PKI-179 at low micromolar concentrations were compared by treating Calu3 cells with MTI-31 , PKI-179 or rapamycin at 1 iM concentration for 1h prior to SARS-CoV-2 infection (MOI 0.01) for 24h.

Interestingly, MTI-31 and PKI-179 displayed a largely superior and unexpected antiviral activity against SARS-CoV-2 in Calu-3 cells compared to rapamycin. While PKI-179 and MTI-31 significantly reduced the amount of viral genome by ~10 times, rapamycin only led to a modest and non-significant antiviral effect against the virus in Calu-3 cells (Fig. 2C).

The superior antiviral action of PKI-179 and MTI-31 against SARS-CoV-2 was confirmed by immunoblotting of the nucleocapsid protein (Fig. 2D). Indeed, while a complete inhibtion in the nucleocapsid viral protein accumulation was observed in cells treated with PKI-179 and MTI-31, only a slight reduction in the same protein accumulation was seen after rapamycin treatment (Fig. 2D). Furthermore, increased LC3B conversion also suggests a superiority for PKI-179 and MTI-31 in mTOR inhibiton and autophagy induction compared to rapamycin.

Altogether, these data indicate that the mTORC inhibitors PKI-179 and MTI-31 strongly suppress SARS-CoV-2 infection at low micromolar concentrations and display significantly better antiviral activity compared to rapamycin at the some dosage.

Example 2 - PKI-179 and MTI-31 inhibit SARS-CoV-2 infection at a post-entry step of the virus replication cycle.

Time-of-addition assays were performed to pinpoint which step of the viral replication cycle is inhibited by PKI-179 and MTI-31. Treatment of VeroE6 hTMPRSS2 and Calu-3 cells with 1 jiM of MTI-31 or PKI-179 was initiated at different time points, after which each drug remained present until the end of the assay. At time 0, cells were infected with SARS-CoV-2, and at 24 hours post-infection, cell lysates were prepared for detection of viral nucleocapsid expression by immunoblotting. Assays were performed with treatments initiated at 1 h prior to infection or at 0, 1 , 2, 4, hours post-infection followed by detection of viral nucleocapsid expression by western blotting 24 hours post-infection (Fig. 3A). The antiviral effect of both drugs was fully retained in VeroE6 hTMPRSS2 and Calu-3 cells when treatment was initiated as late as 4 hours postinfection, as denoted by the equal reduction in viral nucleocapsid protein expression observed at either -1 h or +4h treatment time (Figs. 3B-3C). LC3B conversion and p62 degradation was observed across all timepoints. These data suggest that PKI-179 and MTI-31 inhibit SARS-CoV-2 infection at a post-entry step of the virus replication cycle, and supports that PKI-179 and MTI-31 may find use in both treatment and also prevention of SARS-CoV-2 infections

Example 3 - PKI-179 and MTI-31 decrease SARS-CoV-2 Variants of Concern replication.

To investigate if PKI-179 and MTI-31 were also able to inhibit the infection of SARS- CoV-2 variants of concern (VOC) other than the original strain used throughout the majority of the study (SARS-CoV-2 early pandemic B.1 lineage (FR-4286)), the antiviral efficacy of both drugs against two other VOC, Delta (B.1.617.2) and Omicron (B.1.1.529 BA.1) was tested. Calu-3 cells were treated with PKI-179 and MTI-31 for 1 h prior to infection with either the original strain at a MOI of 0.01 or the Delta (MOI 0.01) and Omicron (MOI 0.1) VOC. At 24 hours post-infection, cells were collected for quantification of viral load by qPCR. PKI-179 and MTI-31 inhibited the infection of the original SARS-CoV-2 and VOC with a similar trend (Figs. 4A-4C).

Example 4 - PKI-179 and MTI-31 are pan-antiviral agents against coronaviruses.

Further, the possible pan-antiviral action of PKI-179 and MTI-31 against other relevant human coronaviruses was tested, including against the SARS-CoV, the Middle East respiratory syndrome coronavirus (MERS-CoV), and the human coronavirus 229E (HCoV-229E). These viruses were used on different cell types as described herein above (see MERS-CoV, SARS-CoV, HCoV-229E infection experiments and TCID50). In all cases, cells were pre-treated with increasing concentrations of PKI-179 or MTI-31 (0.1, 1 and 10 pM) for 1 h prior to challenge with the respective virus at MOI of 0.05. 24 hours post-infection, cells were collected for quantification of viral load by qPCR and supernatants for assessment of viral titers by TCID50. qPCR and TCID50 analyses showed that infection and replication of all viruses tested were inhibited by PKI-179 or MTI-31 at 10 pM (Figs. 5A-5D).

Altogether, these data indicate that PKI-179 and MTI-31 inhibited different coronaviruses in a cell-line independent manner and displayed pan-antiviral action against a range of pathogenic human coronaviruses including SARS-CoV, SARS-CoV- 2, MERS-CoV and HCoV-229E.

Example 5 - PKI-179 and MTI-31 reduce SARS-CoV-2 Infection in primary human airway epithelial cultures.

Further, the antiviral action of PKI-179 and MTI-31 against SARS-CoV-2 was tested in primary human airway epithelial (HAE) cultures derived from healthy donors (Fig. 6A). Here, a pre-treatment of 3h with the different drugs at 10 pM was applied prior to virus infection at MOI 0.1. At 24h post-infection cell lysates were collected, and viral RNA was detected by qPCR (Fig. 6B). Again, PKI-179 and MTI-31 significantly reduced SARS-CoV-2 RNA levels by ~5-fold (Fig. 6C).

Altogether, these results validate the antiviral activity of PKI-179 and MTI-31 against SARS-CoV-2 in a physiologically relevant model of primary HAE. References

G. Li et al., Therapeutic strategies for COVID-19: progress and lessons learned, Nat Rev Drug Discov., 2023, 22, 449-475. https : //do i . org/10.1038/s41573-023-00672-y

N. Dinesh Kumar et al., Moxidectin and Ivermectin Inhibit SARS-CoV-2 Replication in Vero E6 Cells but Not in Human Primary Bronchial Epithelial Cells, Antimicrob Agents Chemother. 2022, 66. https://doi.Org/10.1128/AAC.01543-21

G. Reis et al., Effect of Early Treatment with Ivermectin among Patients with Covid-19, N Engl J Med., 2022, 386, 1721-1731. https://doi.Org/10.1056/NEJMoa2115869

W. H. Self et al., Effect of Hydroxychloroquine on Clinical Status at 14 Days in Hospitalized Patients With COVID-19: A Randomized Clinical Trial, JAMA, 2020, 324, 2165-2176. https://doi.org/10.1001/jama.2020.22240

A. H. Williams & C. G. Zhan, Staying Ahead of the Game: How SARS-CoV-2 has Accelerated the Application of Machine Learning in Pandemic Management, BioDrugs, 2023, 37, 649-674. https://doi.Org/10.1007/S40259-023-00611 -8

S. Matsuyama et al., Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells, Proc Natl Acad Sci, 2020, 117, 7001-7003. https://d0i.0rg/l 0.1073/pnas.2002589117

M. E. Carter-Timofte et al., Antiviral Potential of the Antimicrobial Drug Atovaquone against SARS-CoV-2 and Emerging Variants of Concern, ACS Infect Dis, 2021 , 7, 3034-3051. https://doi.org/10.1021/acsinfecdis.1c00278 D. Olagnier et al., SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate, Nat Commun., 2020, 11(1):4938. https ://do i . org/10.1038/s41467-020- 18764-3

V. S. Raj et al., Dipeptidyl peptidase 4 is a functional receptor for the emerging himan coronavirus-EMC, Nature, 2013, 495, 251-254. https://doi.Org/10.1038/nature12005

L. Vijgen et al., Development of One-Step, Real-Time, Quantitative Rverse Transcriptase PCR Assays for Absolute Quantitation of Human Coronaviruses OC43 and 229E, J Clin Microbiol., 2005, 43(11). https://doi.Org/10.1128/JCM.43.11.5452-5456.2005

V. M. Corman et al., Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT- PCR, Euro Surveill. 2020, 25(3), 23-30. https://doi.Org/10.2807/1560-7917.ES.2020.25.3.2000045

P. J. Mullen et al., SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORCI inhibition, Nat Commun, 2021 , 12, 1876. https : //do i . org/10.1038/s41467-021 -22166-4

Claims

1 . A pharmaceutical composition consisting of: a. one or more small-molecule drugs which is i) a PI3K inhibitor and/or blocker and/or ii) a mTOR inhibitor and/or blocker; and b. optionally, one or more therapeutically inactive excipients; for use in a method of treatment, prevention and/or amelioration of a viral disease or infection, with the proviso that the small-molecule inhibitor is not rapamycin.

2. The composition for use of any one of the preceding claims, wherein said viral disease or infection is caused by a virus selected from the group consisting of coronavirus, adenovirus, picornavirus, flavivirus, herpesvirus, filovirus, poxvirus and retrovirus.

3. The composition for use of any one of the preceding claims, wherein said viral disease or infection is caused by coronavirus.

4. The composition for use of any one of claims 2 to 3, wherein the type of coronavirus is selected from the group consisting of SARS-CoV-2, MERS-CoV, SARS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1.

5. The composition for use of any one of claims 2 to 4, wherein the type of coronavirus is selected from the group consisting of SARS-CoV-2, MERS-CoV, and HCoV-229E.

6. The composition for use of any one of claims 2 to 5, wherein the type of coronavirus is SARS-CoV-2.

7. The composition for use of any one of claims 4 to 6, wherein said SARS-CoV-2 virus is characterized by a lineage and/or variant selected from wild-type, alpha, beta, delta or omicron.

8. The composition for use of claim 7, wherein the variant is omicron.

9. The composition for use of claim 8, wherein the omicron variant is a virus of B.1.1.529 lineage, such as B.1.1.529 or B.1.1.529.1 (BA.1).

10. The composition for use of claim 7, wherein the variant is delta, optionally, wherein the delta variant is a virus of B.1617.2 lineage.

11 . The composition for use of claim 7, wherein the variant is beta, optionally, wherein the beta variant is a virus of B.1.351.1 lineage.

12. The composition for use of any one of the preceding claims, wherein the smallmolecule drug is a PI3K inhibitor and/or blocker.

13. The composition for use of any one of the preceding claims, wherein the smallmolecule drug is a mTOR inhibitor and/or blocker.

14. The composition for use of any one of the preceding claims, wherein the smallmolecule drug is a dual PI3K/mTOR inhibitor and/or blocker.

15. The composition for use of any one of the preceding claims, wherein the smallmolecule drug is one or more selected from the group consisting of or a pharmaceutically acceptable prodrug, ester, salt or hydrate thereof

16. The composition for use according to any one of the preceding claims, wherein the small-molecule drug is PKI-179 and/or MTI-31 or a pharmaceutically acceptable prodrug, ester, salt or hydrate thereof.

17. The composition for use according to any one of claims 1 to 16, wherein the small-molecule drug is or a pharmaceutically acceptable prodrug, ester, salt or hydrate thereof.

18. The composition for use according to any one of claims 1 to 16, wherein the small-molecule drug is or a pharmaceutically acceptable prodrug, ester, salt or hydrate thereof.

19. The composition for use of any one of the preceding claims, wherein the smallmolecule drug is characterized by an IC50 of 50 pm or less, such as 40 pm or less, such as 30 pm or less, such as 25 pm or less, such as 20 pm or less, such as 10 pm or less, such as 5 pm or less, such as 1 pm or less, such as 0.5 pm or less, in particular wherein the IC50 is evaluated against SARS-CoV-2.

20. The composition for use of any one of the preceding claims, wherein the composition does not comprise a compound of any one of formula (I) or formula (II) as defined in WO2022/219157.

21. The composition for use according to any one of the preceding claims, wherein the composition is formulated for parenteral administration, such as intravenous, subcutaneous or intramuscular; or for enteral administration such as oral, rectal or nasogastric; or for topical administration such as pulmonary, intranasal, or intratracheal.

22. The composition for use according to any one of the preceding claims, wherein said composition is to be administered in a dosage of from 1 pg/kg -10,000 pg/kg body weight, such as 1 pg/kg - 7,500 pg/kg, such as 1 pg/kg - 5,000 pg/kg, such as 1 pg/kg - 2,000 pg/kg, such as 1 pg/kg - 1 ,000 pg/kg, such as 1 pg/kg - 700 pg/kg, such as 5 pg/kg - 500 pg/kg, such as 10 pg/kg to 100 pg/kg bodyweight.

23. The composition for use according to any of the preceding claims, wherein said administration is repeated daily.

24. The composition for use according to any one of claims 1 to 22, wherein said administration is repeated at least 1-3 times weekly, such as 2-5 times weekly, such as 3-6 times weekly.

25. The composition for use according to any one of claims 1 to 23, wherein said administration is repeated 1 to 8 times daily, such as 2 to 5 times daily.

26. The composition for use according to any one of the preceding claims, wherein said use is for a method of treatment of a subject having an immunodeficiency.

27. A pharmaceutical composition comprising a combination of PKI-179 and MTI-31 for use as a medicament.

28. A pharmaceutical composition comprising a combination of PKI-179 and MTI-31 for use in a method of treatment, prevention and/or amelioration of a viral disease or infection.

29. The composition for use according to claim 28, wherein said viral disease or infection is caused by a coronavirus.

30. The composition for use according to any one of claims 28 to 29, wherein said viral disease or infection is caused by SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E or a subvariant of any of the foregoing.

31. The composition for use according to any one of claims 28 to 30, wherein said viral disease or infection is caused by SARS-CoV-2, SARS-CoV, MERS-CoV or HCoV-229E, preferably SARS-CoV-2.