Nature's hope of curing dengue is rekindled—a pan-serotype dengue inhibitor targeting NS3-NS5 interaction

Written by Wangye | XI If you continue to pay attention to the global new crown epidemic, you may know that as of October 8, 2021, the number of confirmed patients worldwide has reached 236599025 (https://covid19.
who.
int/)
.

What you may not know is that there are still about 100-400 million people infected with Dengue virus each year, and it is estimated that there are 96 million patients with symptoms alone
.

Dengue virus is a flavivirus transmitted by mosquito bites.
Its main vectors are Aedes aegypti and A.
albopictus.
They are also chikungunya virus, yellow fever virus and The transmission vector of Zika virus
.

In the past 50 years, the dengue virus infection rate has increased by 30 times.
At present, dengue virus infection has occurred in 128 countries and regions around the world, and 3.
9 billion people are under the threat of the virus
.

There are four serotypes of dengue virus (DENV-1~4).
The infection of one serotype may aggravate the chance of infection of other serotypes.
This is due to the low or medium-efficiency antibodies produced in the patient's body to promote subsequent serotypes Virus infection, this phenomenon is called the ADE effect
.

At present, a vaccine called Dengvaxia has been approved in some countries for the prevention of individuals over nine years of age, but it is only recommended for patients with a history of dengue exposure
.

However, therapeutic drugs have not yet been approved.
The difficulty in development is that the drugs should be suitable for oral administration, can quickly reduce the viral load to prevent the progression of the disease to severe disease, and are effective in dealing with the four serotypes
.

Despite the difficulties, the process of drug development has never stopped.
On October 6, 2021, the Belgian scientist Marnix Van Loock and the team of Johan Neyts published a research article titled A pan-serotype dengue virus inhibitor targeting the NS3–NS4B interaction in Nature.
Announced that an inhibitor named JNJ-A07 can inhibit 21 dengue virus clinical strains of 4 serotypes at nanomolar to picomolar concentrations; this molecule is not easy to produce drug-resistant mutations, and targets the blocking viruses NS3 and NS4B To prevent the formation of viral replication complexes; mouse experiments show that it has excellent pharmacokinetic characteristics and good safety, can play a preventive effect, and is also effective when delayed administration
.

Analogs of this molecule have entered subsequent development
.

The JNJ-A07 class of molecules is not the first to appear.
In fact, the team has reported the synthesis and testing of its lead compounds in 2018: through in vitro cell experiments, a new indole compound was screened out of about 2000 drug candidates.
Effectively inhibit the infection of cells by the DENV-II strain
.

In this report, the researchers first confirmed that JNJ-A07 can exert antiviral effects at nanomolar to picomolar concentrations in various cell lines such as Huh7, Vero, and C6/36, especially in immature dendritic cells.
It is equally effective in primary cells, which are considered to be the primary target cells for virus invasion
.

The author also found that the median effective concentration (EC50) of JNJ-A07 on 21 dengue virus strains of 4 serotypes reached the nanomolar to picomolar level.
These 21 clinical strains cover all currently known dengue virus genes Type, and JNJ-A07 has no inhibitory effect on other flaviviruses, so it is a specific inhibitor of dengue virus
.

Next is to find the target
.

The author found that as long as the virus RNA synthesis has not yet started or has not reached the detection line, even if the addition of JNJ-A07 to the infected cells is delayed, the antiviral activity is not significantly weakened; and once the viral RNA is synthesized, the antiviral activity gradually becomes Lost
.

Therefore, the role of JNJ-A07 points to the viral RNA synthesis machine
.

In order to determine the target, the authors conducted a screening experiment for drug-resistant mutant strains and found that in the continuous subculture with the addition of the compound, the resistance mutation of DENV-2 to JNJ-A07 could not be detected until the 15th week, and the complete mutant strain was not detected until the 15th week.
It only appeared after 40 weeks
.

After sequencing, it was found that the mutation resulted in the substitution of three amino acids in the virus NS4B gene (in two parallel experiments, one group had mutations of L94F, T108I, and T216N; the other group had mutations of V91A, L94F, and T108I, and some of them showed F47Y, P104S, T216P mutation), on the one hand, it shows that NS4B is the target, on the other hand, it also shows that the resistance threshold of JNJ-A07 is very high
.

Subsequent experiments also found that mutation sites in human-derived cell lines can prevent the virus from replicating in mosquito cells.
In other words, even if the virus produces drug-resistant mutations during drug treatment, the mutant strains almost lose the ability to continue to be transmitted through mosquito bites.
Possible
.

Dengue virus NS4B protein can induce the production of endoplasmic reticulum-derived membrane vesicles, which is where the virus replication occurs; NS4B can also block the IFN-α/β pathway to prevent the establishment of host cell antiviral response
.

The encoded protein of flavivirus is first translated into a long polypeptide chain, which is cleaved and matured by the host and viral protease; among them, NS4B needs to be processed from the NS4A-2K-NS4B precursor, and the viral protease-helicase complex NS2B-NS3 participates in this Process
.

So the author tested the effect of JNJ-A07 on the NS3-NS4B interaction, and used the NS4B specific pull-down experiment, and found that JNJ-A07 can inhibit 95% of the NS4B-NS3 interaction at a 45-fold EC50 concentration; When V91A, L94F, T108I, T216N mutations occur, JNJ-A07 almost no longer affects the NS4B-NS3 interaction
.

Further experiments also found that mutants V91A and T108I enhanced the NS3-NS4B interaction in a dose-dependent manner
.

What is the role of JNJ-A07 in the body? The author first verified that it has good pharmacokinetic characteristics in mice and rats, and it has no side effects when administered orally at a dose of 300mg/Kg for 15 days
.

Then, using the infection model AG129 mouse to challenge the DENV-2 type RL strain, through oral administration twice a day, it was found that JNJ-A07 can significantly inhibit the viral load in the spleen, kidney, liver and other organs in a dose-dependent manner.
The levels of IL-18, IFN-γ, TNF, IL-6 and other inflammatory factors are close to normal
.

Next, the researchers tested the protective effect of JNJ-A07 on mice challenged with a lethal dose of the virus (106 PFU) and found that when administered at a dose of 30 mg/kg, the survival rate of the mice reached 90%, even if it was administered at 1 mg/kg.
The survival rate can still reach 75%
.

At non-lethal doses, 30, 10, and 3 mg/kg administration can maintain viral RNA near the detection line
.

Finally, the authors found that JNJ-A07 has both preventive and therapeutic effects, and it still produces antiviral effects quickly after 4-5 days of non-lethal dose of virus infection
.

Therefore, JNJ-A07 shows excellent antiviral activity in mice
.

The current experimental results show that JNJ-A07 is a highly effective, pan-serotype dengue virus inhibitor, targets NS4B, is not easy to produce drug-resistant mutations, and shows good pharmacokinetic characteristics and safety in mice.
Effectively prevent and treat dengue virus infection, showing promising development prospects
.

Nature magazine also published a point of view article A step towards dengue therapeutics written by Scott B.
Biering and Eva Harris of the University of California, Berkeley, introducing the conclusions of the study and looking forward to its subsequent development.
At the same time, it pointed out the specific mechanism of JNJ-A07 inhibiting NS4B.
It is subject to structural biology research, whether it can be combined with other antiviral drugs and other issues still need to be further studied and confirmed
.

Can JNJ-A07 analogues achieve ideal results in subsequent development? We will wait and see
.

(Simplified diagram of the mechanism of JNJ-A07 inhibiting the replication of dengue virus) Original link: https://doi.
org/10.
1038/s41586-021-03990-6 Platemaker: 11 References 1, https://www.
who.
int/news-room/fact-sheets/detail/dengue-and-severe-dengue.
2, Bardiot, D.
et al.
Discovery of indole derivatives as novel and potent dengue virus inhibitors.
J.
Med.
Chem.
61 , 8390–8401 (2018).
3, Płaszczyca, A.
et al.
A novel interaction between dengue virus nonstructural protein 1 and the NS4A-2K–4B precursor is required for viral RNA replication but not for formation of the membranous replication organelle.
PLoS Pathog.
15, e10077362019 (2019).
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