Development Of Broad-Spectrum Antiviral Inhibitors To Target Pathogens Of Pandemic Concern

1. Abstract

Small molecule, orally bioavailable, broad-spectrum antivirals are critical for stopping the next outbreak or pandemic that occurs. Current drugs have a number of issues including lack of oral bioavailability (remdesivir), serious drug-drug interactions (Paxlovid), and funds for pandemic preparedness are now limited as NIH ended the AViDD (AntiVIral Drug Discovery centers) after year 3 (rather than the initially promised 5 years) due to Congress and NIH severely cutting support for pandemic preparedness research. This is likely due to a naive “head in the sand” mentality, however it is foolish to believe we will never have another outbreak and in fact we are indeed currently in the middle of a hantavirus outbreak that has caused panic, the largest Ebola outbreak to date in Africa, as well as the widespread measles outbreaks in the USA. Our molecules have exhibited potent broad-spectrum activity across a number of critical viral families, they have exhibited no toxicity, and they appear to work by a different mechanism than typical nucleoside drugs. This makes them ideal for use in combination therapy. We are proposing to carry out mechanism of action studies as well as to improve upon their activity in this proposal so that we can obtain the necessary data needed to attract licensing agreements and further funding. We fell this can be achieved in the next 9-12 months.

2. Introduction

Introduction: Small molecule, orally bioavailable, broad-spectrum antivirals are critical for stopping the next outbreak or pandemic that occurs. Current drugs have a number of issues including lack of oral bioavailability (remdesivir), serious drug-drug interactions (Paxlovid), and funds for pandemic preparedness are now limited as NIH ended the AViDD (AntiVIral Drug Discovery centers) after year 3 (rather than the initially promised 5 years) due to Congress and NIH severely cutting support for pandemic preparedness research.^1-3 This is likely due to a naive “head in the sand” mentality, however it is foolish to believe we will never have another outbreak and in fact we are indeed currently in the middle of a hantavirus outbreak that has caused panic, the largest Ebola outbreak to date in Africa, as well as the widespread measles outbreaks in the USA.^4-6

In that regard, nucleoside analogues are one of the most important classes of drugs on the market due to their ability to mimic the naturally occurring nucleosides found in our DNA and RNA. They typically work by getting incorporated by the viral polymerases into the growing nucleoside chain and causing chain termination.⁷ The Seley-Radtke group has synthesized fleximer nucleoside analogues which have several advantages as therapeutics against RNA viruses. A number of our fleximer analogues (1) have exhibited broad-spectrum activity across several viral families; (2) they are orally bioavailable; (3) they have exhibited efficacy in the nanomolar and single digit micromolar ranges; (4) they have exhibited little to no host toxicity, and (5) importantly, they do not inhibit the viral polymerases like most nucleoside analogues, but appear to be working by inhibition of viral methyltransferases (MTases).^8-12 This would make them ideal for use in combination therapy.

Of the 160+ nucleoside analogues developed in our lab in the past three years under the auspices of the NIH U19 AVIDD program (UNC-CH), we identified the top three compounds that formed the basis for this project (Figure 1).^8-10 The fleximers offer forth several strategic advantages over conventional nucleoside analogues: i) They can overcome point mutations in an enzyme binding site in resistant strains of a virus by engaging secondary amino acid residues not previously involved in the mechanism of action (MOA), allowing them to retain their full activity in the face of viral resistance mechanisms; ii) They are able to increase binding affinity by engaging better binding partners; and iii) They exhibit potent activity against viruses that the parent nucleoside has no activity against; e.g. our fleximers related to Acyclovir, a nucleoside analogue approved for treating herpes virus.^13-15 Several of the acyclic fleximers (Flex-acyclovir analogues) exhibited low nanomolar, and single digit micromolar levels of activity against coronaviruses such as MERS and SARS-2, flaviviruses such as Dengue (DENV), Yellow Fever (YFV), West Nile (WNV), and Tickborne encephalitis (TBEV), as well as filoviruses such as Ebola, Sudan and Marburg.^15-17 Some were tested in mice up to 300 mg/kg with no adverse effects, while acyclovir exhibits zero activity against these viruses even at doses up to 1000 mM/kg.

As shown above in Figure 1, the three lead compounds have shown broad-spectrum activity against several different viral families. Most recently, KAD-039 was screened against additional flaviviruses and as shown in the Table below, they exhibited activity against WNV, ZIKV, JEV, TBEV, POWV, and JFDV.^{9, 18}

Virus	Strain	KAD-039
Virus	Strain	Huh-7	A549
WNV	Eg-101	4.31 ± 0.99	6.54 ± 0.65
ZIKV	Paraiba01	4.18 ± 2.84	10.74 ± 2.84
JEV	P3	2.13 ± 0.20	9.10 ± 1.62
TBEV	Hypr	11.27 ± 5.04	9.49 ± 2.10
POWV	LB	1.77 ± 0.58	8.24 ± 0.59
KFDV	W-377	2.69 ± 1.20	6.14 ± 0.43
LGTV	TP21	1.04 ± 0.12	4.61 ± 1.57
LIV	LI/31	7.66 ± 0.42	21.04 ± 0.4

Reported values correspond to EC₅₀ and are given in µM.

The compounds are also being sent (through the NIH/NIAID testing mechanism) to Utah to be tested against various bunyaviruses and other viruses including Andes, Junin, Rift Valley Fever virus, Oropouche and Heartland. They will also be sent via our collaboration with Dr. Brian Gowen (Utah), a leading expert in hanta- and bunyaviruses. Given the broad range of viral families against which the fleximers have already shown activity, we anticipate that these compounds may exhibit activity against these viruses as well.

Hypotheses

Hypothesis: As mentioned above, the fleximers are not recognized and incorporated by the viral RdRps but appear to be inhibiting the N7 viral MTases and/or capping mechanism.^{14, 16} As such, they will be ideal in combination therapies with drugs that work by other mechanisms. In that regard, we recently provided the parent compound and the prodrug of KAD-039 to our collaborators (Bruno Canard and his team, one of the world’s leading experts in nucleoside metabolism, MTases, and elucidating MOA) in Marseille for MOA studies in the MTases. They will also use enzymatic methods to synthesize the corresponding triphosphate (TP), as we have had difficulties making it chemically. Computational modeling by one of our collaborators at Emory University is shown below in Figure 2.¹⁸ These computational findings support this hypothesis as they fit perfectly within the GTP-binding pocket of the viral MTases, but not in the human, which explains their selectivity and lack of toxicity. In addition, the modeling has shown increased hydrogen bonding interactions with the azido moiety on KAD-039, which appears to be adding to its efficacy.

Materials and Procedures

Materials and Procedures: The aims of this project will be to (1) further evaluate the fleximers potential for activity against new viral families, including bunyaviruses such as hantavirus, (2) conduct MOA and metabolism studies, (3) perform additional computational studies to see if we can improve upon the activity, (4) do PK/ADME studies, then (5) advance the most promising candidate to animal studies to obtain maximum tolerated dose (MTD), efficacy, and challenge studies. We realize that the funding available through this mechanism is unlikely to support animal studies, but it is our hope that the mechanistic and other results we obtain through this grant will help us obtain additional funding and/or attract licensing agreements.

Synthesis: Prof. Seley-Radtke has over 37 years of experience in the design and synthesis of nucleoside/nucleotide analogues, thus we don’t anticipate any issues with pursuing any new analogues that the modeling suggests. Currently the group is working on synthesizing several analogues of CEM-057 with different prodrug moieties given their proven activity on the orally bioavailable version of Remdesivir. In addition, although we have had some difficulties in the synthesis of the KAD-039-TP chemically, we are pursuing two different enzymatic routes including one through our collaborators in Marseille. It should be noted that we routinely make triphosphates of other fleximers so we believe this will not be a problem for any other analogues.^{8, 16} We will follow our standard procedures as well as any new literature as needed.

Modeling: Prof. Stefan Serafianos’s modeler is Dr. Ryan Slack – he generated all of the figures shown above and is an expert in the field. He will use his standard programs and procedures, which can be found in his publications, as well as our invited publication that is under review at ChemMedChem.^18-21

Antiviral testing: All of our collaborators use standard protocols, controls, and at least three runs to establish standard deviations, that have been published in top peer reviewed journals.^{8, 9} The NIH testing protocols are online. Given the space limitations, we won’t elaborate on specifics here since they employ many different contractors to carry out various screenings.^{8, 9, 18, 22-29}

MTase MOA and metabolism testing: Prof. Canard has long studied a number of FDA approved nucleoside analogues’ MOA for their activity against viral RdRps, viral MTases, as well as how the McGuigan ProTide moieties are metabolized to provide the monophosphate necessary for conversion to the biologically active triphosphate.^30-36 He has published some of the leading papers on the MOA for Remdesivir, AT-527.^{30, 37-39}

ADME/PK: We (as well as many others including Gilead Pharmaceuticals) have used the UMinn Center for Drug Design several times for previous ADME/PK studies and they too have well-established protocols.

Planned Analyses

Based on the outcome of antiviral testing, modeling and mechanistic studies we will proceed to PK/ADME studies with the top 1-2 compounds. All testing is done in at least triplicates to establish appropriate standard deviations.

Ethics and Data Management

There are no ethical concerns for this project and the data will be managed by Prof. Seley-Radtke and her collaborators.

4. Pilot Data (Optional)

See above findings.

5. Budget

Budget: $7500 for chemicals, reagents and chromatography supplies.

$2000 for NMR/HRMS and elemental analysis.

$500 for fedex to send samples to collaborators and for elementals.

6. References

References:

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Development Of Broad-Spectrum Antiviral Inhibitors To Target Pathogens Of Pandemic Concern

Development Of Broad-Spectrum Antiviral Inhibitors To Target Pathogens Of Pandemic Concern

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