8 Nov Episode 82 Polymerase inhibitors and human protease inhibitors

Sun, 11/08/2020 - 20:20

Dear colleagues,

Upon request, I focused on the background and present status of two possible “repurpose” treatment options: Truvada (Tenofovir-Emtricitabine) and Camostat. I broadened the scope to the class of these compounds: resp nucleos(t)ide polymerase inhibitors and host protease inhibitors and I added also links to the ongoing corresponding trials at ClinicalTrials.gov

As you will see, the published evidence is mainly “circumstantial” and largely limited to in vitro data, with sometimes contradictory results (which may be because of use of different cell lines or techniques). Since quite a few clinical trials are ongoing, we may have more substantial evidence soon.

Tenofovir and other polymerase inhibitors

Ref Nuc 1 COVID-19 in PLWH on ART (Del Amo Ann Int Med)

The risk for COVID-19 diagnosis is not higher in HIV-positive persons than in the general population,
HIV-positive patients receiving TDF/FTC had a lower risk for COVID-19 and related hospitalization than other HIV-positive persons.

Ref Nuc 2 Molecular docking study (Ref Nuc 2 Elfiky Life Sciences)

Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir tightly bind to SARS-CoV2 RdRp.
In addition, guanosine derivative (IDX-184), Setrobuvir, and YAK as top seeds for antiviral treatments of SARS-CoV-2 strain specifically.

Ref Nuc 3 Tenofovir and Emtricitabine Triphosphates are inhibitors of SARS-CoV-2 RdRP (Jockush bioRxiv April 2020):

This is shown by in vitro biochemical analysis of the “RNA primer extension” by the purified RNA-dependent RNA polymerase complex. So no effect on real virus is shown.

Ref Nuc 4 and 5: A library of nucleotide analogues terminate RNA synthesis of SARS-CoV-2 (Jockush Antiv. Res) and Nucleotide Analogues as Inhibitors of SARS-CoV‑2 Polymerase (Chien J Proteome):

Using the same technology, these authors also showed a similar in vitro activity by the tri-phosphate forms of other established nucleoside analogues Zidovudine, Lamivudine, Abacavir, Tenofovir-Alafenamide, Sofosbuvir, Carbovir, Ganciclovir, Stavudine and Entecavir and of the experimental compounds OMe-UTP and Biotin-16-dUTP ,

Ref Nuc 6 and 7: Sofosbuvir has been shown to inhibit SARS-CoV-2 replication in Huh-2 (human hepatoma-derived) and Calu-3 (Type II pneumocyte-derived) cells with EC50 values of 6.2 and 9.5 μM, respectively, but not in Vero-E6 cells (Sacramento bioRxiv Oct 2020). Sofosbuvir was also shown to protect human brain organoids from infection by SARS-CoV-2 (Mesci bioRxiv May 2020).

Ref Nuc 8: A nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of antiinfective drugs for COVID-19 (

In this assay a modified virus is used that emits light when it infects an ACE-2 transfected A549 cell line. It is shown that Remdesivir and Chloroquine are active in the low µM range with a high selectivity index (284 and > 38).

However, nelfinavir, rupintrivir, and cobicistat as very active compounds (EC50 around 1 µM), but a rather low selectivity index.

Remarkably also, Favipriravir, Sofosbuvir, Tenofovir and Emtricibatine were not active up to 10 µM.

Ref Nuc 9: Antiviral efficacy in ferrets (Park mBio):

Lopinavir-ritonavir-, hydroxychloroquine sulfate-, or emtricitabine-tenofovir marginally reduced the overall clinical scores of infected ferrets but did not significantly affect in vivo virus titers.

Host Protease inhibitors (HPI)

At least three endogenous proteases (TMPRSS2, Cathepsin L and furin) have a role in the entry process: activation of spike for efficient interaction with ACE2 receptor and endosomal fusion.

The putative TMPRSS2 inhibitors Camostat mesylate and nafamostat mesylate used for treatment of chronic pancreatitis

Ref HPI 1: Hoffman paper in Cell (April 2020): see Fig 4 p.7-8: Camostat in µM concentrations alone can inhibit entry in some cell lines by inhibiting TMPRSS2, but in others a combination with E64 (a Cathepsin B/L inhibitor) is needed.

Ref HPI 2: Hofmann in AAC shows that nafamostat mesylate blocked SARS-CoV-2 infection of human lung cells with markedly higher efficiency than camostat mesylate

Ref HPI 3: Tianxiao Liu in Pharmacology & Therapeutics 213 (2020) hypothesize that combined use of TMPRSS2 inhibitor camostat mesylate or nafamostat mesylate and a Cathepsin L inhibitor will blunt substantially coronavirus infection of human cells.

The authors propose the following benefits of this combination versus SARS-CoV-2 (see Fig 2 and 3):

Cathepsin L inhibitor + TMPRSS2 inhibitor Camostat will inhibit entry (no “activation” of the spike)
CatL inhibitors will also inhibit generation of fusion peptide in the Spike and therefor block the release of genomic RNA from the endosome into cytoplasma
CatL is redundant for antigen presentation, T and B cell function, can be replaced by CatS.
A concern remains that chronic use of CatL inhibitors have been associated with pulkmonary fibrosis

Ref HPI 4: Bestle in Life Science Alliance shows that both TMPRSS2 and Furin are essential for proteolytic activation of the spike. A combination of specific inhibitors has a synergistic effect.

Ref HPI 5: Cheng et al in Cell Reports shows that various furin inhibitors block SARS-CoV-2 spike activation and suppress viral replication and syncytium induction in cell lines

Ref HPI 6: Wu in iScience performed a large in silico and biochemlical screening on 4000 potential furin inhibitors and found that the anti-parasitic drug Diminazene was able to inhibit the furin protease activity with EC50 of 5.4 µM.

Ref HPI 7: It was suggested that the commonly used mucolytic agent bromhexine could also be used, because it inhibits TMPRSS2 (Depfenhart Int Emerg Med 2020).

Ref HPI 8: A surprisingly favorable effect of bromhexine was obtained in an Iranian open randomized-controlled study in 78 patients. The patients with standard treatment + bromhexine vs standard alone had less ICU admission (2 vs 11), less intubation (1 vs 9) and less mortality (0 vs 5), with also beneficial effects on secondary outcomes such as fever, cough, dyspnea, but also CRP and LDH (which were all similar in both groups at study entry)…..

Ongoing clinical trials on ClinicalTrials.gov

Truvada: 1 study

https://clinicaltrials.gov/ct2/results?cond=Covid19&term=truvada&type=&rslt=&age_v=&gndr=&intr=&titles=&outc=&spons=&lead=&id=&cntry=&state=&city=&dist=&locn=&rsub=&strd_s=&strd_e=&prcd_s=&prcd_e=&sfpd_s=&sfpd_e=&rfpd_s=&rfpd_e=&lupd_s=&lupd_e=&sort=

Emtricitabine/Tenofovir Alafenamide: 1 study

https://clinicaltrials.gov/ct2/results?cond=Covid19&term=Alafenamide&type=Intr&rslt=&age_v=&gndr=&intr=&titles=&outc=&spons=&lead=&id=&cntry=&state=&city=&dist=&locn=&rsub=&strd_s=&strd_e=&prcd_s=&prcd_e=&sfpd_s=&sfpd_e=&rfpd_s=&rfpd_e=&lupd_s=&lupd_e=&sort=

Sofosbuvir: 9 trials

https://clinicaltrials.gov/ct2/show/NCT04530422?term=Sofosbuvir&type=Intr&cond=Covid19&draw=2&rank=1

Favipiravir: 39 trials

https://clinicaltrials.gov/ct2/results?cond=Covid19&term=favipiravir&type=Intr&rslt=&age_v=&gndr=&intr=&titles=&outc=&spons=&lead=&id=&cntry=&state=&city=&dist=&locn=&rsub=&strd_s=&strd_e=&prcd_s=&prcd_e=&sfpd_s=&sfpd_e=&rfpd_s=&rfpd_e=&lupd_s=&lupd_e=&sort=

Camostat: 13 trials https://clinicaltrials.gov/ct2/results?cond=Covid19&term=camostat&type=Intr&rslt=&age_v=&gndr=&intr=&titles=&outc=&spons=&lead=&id=&cntry=&state=&city=&dist=&locn=&rsub=&strd_s=&strd_e=&prcd_s=&prcd_e=&sfpd_s=&sfpd_e=&rfpd_s=&rfpd_e=&lupd_s=&lupd_e=&sort=

Nafamostat: 4 trials

https://clinicaltrials.gov/ct2/results?cond=Covid19&term=nafamostat&type=Intr&rslt=&age_v=&gndr=&intr=&titles=&outc=&spons=&lead=&id=&cntry=&state=&city=&dist=&locn=&rsub=&strd_s=&strd_e=&prcd_s=&prcd_e=&sfpd_s=&sfpd_e=&rfpd_s=&rfpd_e=&lupd_s=&lupd_e=&sort=