INDENTIFYING DRUG TARGETS

Fig. 1.  Influenza virus

Structure of
Influenza Virus

It is essential to understand the structure and lifecycle of a virus before discussing its drug targets. Flu is caused by the influenza virus. It has eight segments of RNA that vary between 934 and 2,341 nucleotides in length. In comparison, the human DNA has over 3 billion nucleotides on each strand. The virus is enveloped in a membrane over a structural protein mantle. About 400 hemagglutinin (H) and 100 neuraminidase (N) proteins are anchored in the envelope. There are four groups of the virus (Types A-D), 18 variations of H (H1-H18) and 11 variations of N (N1-N11). Only Types A and B affect humans. An influenza virus is identified by its unique H and N. For instance, the 2009 Swine Flu was caused by a sudden and extensive change in A(H1N1), which is a Type A virus carrying H1 and N1. A virus never has multiple types of H or N on its surface.

Fig. 2.  Lifecycle of influenza virus (doi.org/10.1016/j.tips.2007.04.005)

Lifecycle of
Influenza Virus

A virus has to get inside a cell to replicate and survive. All viruses carry an appendage on their surface that is considered harmless by the cells. In case of influenza it is hemagglutinin, which is a naturally occurring protein in humans. The cells in our respiratory tract have sialic acid on their surface that binds naturally to hemagglutinin. When we breath in a virus, it touches the sialic acid on the cell with its hemagglutinin appendage (Step 1 in Fig. 2). Sensing only the hemagglutinin, the cell is tricked into believing that the virus is a harmless nutritional molecule and pulls in the entire virus (Step 2). It realizes its mistake after engulfing the virus and tries to kill it with lysosome. Lysosomes are extremely acidic (pH = 4.5-5.0) organelles in the cytoplasm. Unfortunately, the lysosomes manage to destroy only the outer envelope of the virus (Step 3). That releases the viral RNA which quickly moves to the nucleus and uses the cellular machinery to make hundreds of copies of itself (Step 4). Mature viruses move towards the cell surface for exit. Neuraminidase, the other characteristic protein of influenza virus, plays a key role at this stage. The mature virus tries to bud out of the cell membrane, but it is not strong enough to extricate itself from the elastic membrane (Step 5). Neuraminidase snips and releases the bud. In the process, the virus grabs a piece of cell membrane to cover itself (Step 6). Therefore, the eight segments of the influenza RNA code only eight proteins that are unique to it including the structural protein. It uses the cellular material and replication apparatus for everything else. As mentioned in the Section Biochemistry of Viruses, -ases are enzymes (i.e. protein-based catalysts), so is neuraminidase. Viruses leaving the cell infect neighboring cells or are released into the air through coughing, sneezing and breathing.

Drug Targets
Against
Influenza Virus

After reviewing the structure and lifecycle of the influenza virus, we are ready to discuss the potential drug targets against the virus. Target selection is all about identifying the stages in lifecycle when a microbe is accessible and vulnerable. The candidate targets are noted in the beige boxes in Figure 2. For example, as the name implies, RNA polymerase inhibitor blocks the RNA polymerases. As noted in the Section Biochemistry of Viruses, polymerases catalyze the replication of genetic material. Influenza needs its own version of RNA polymerase. So, it packs the necessary equipment. One of the eight strands of influenza RNA codes for this polymerase. However, not all potential drug targets are viable. In principle, an RNA polymerase inhibitor will block the formation of genetic material for the progeny viruses. But that could also disrupt our native RNA polymerases and RNA production. So far, the pharmaceutical scientists have attained success with only neuraminidase inhibitors, primarily because they work outside the cell. It is hard to hit targets inside the cells because of the cell's fortification at the membrane boundary and overlapping function such as RNA polymerases. Oseltamivir (tradename Tamiflu) and zanamivir (tradename Relenza) are blockbuster neuraminidase inhibitors. In fact, the inhibitors are so effective that they kill over 99% of all influenza strains. In comparison, vaccines are extremely selective in targeting a strain, which is a double-edge sword.

Vaccines
Against
Influenza Virus

Vaccines work by fortifying our immune system against future invasion by microbes. It has been discussed in greater detail in the Section Viral Strains and Immunity. The immune system operates outside the cells, e.g. in blood stream and lymphatic system. Identifying a foreign material is the first and most critical part of immune defense. As seen in Figure 1, membrane, hemagglutinin and neuraminidase are the three most visible parts of the influenza virus. Since the newly formed virus grabs a part of the cellular membrane on its way out, the immune system cannot distinguish between the membranes of the virus and cells. That leaves hemagglutinin and neuraminidase as the two choices. For reasons not well understood, our immune system reacts much more to a foreign hemagglutinin than to a neuraminidase. Therefore, all flu vaccines are built around hemagglutinin.

Drugs or
Vaccines?

The short answer is, both. Drugs and vaccines complement each other is fighting diseases. There are numerous pros and cons for both. Drugs, vaccines and surgery are the three common means for treating diseases. Vaccine is the most cost-effective method among those, serving large populations. Vaccination is about prevention, thereby avoiding the danger, cost and misery of falling sick. The herd immunity is another great benefit. Smallpox and polio were "eliminated" through herd immunity. Those viruses still exist on Earth but not in sufficient quantity so as to be a threat. The importance of vaccines is evident from the current race to immunize against COVID-19. On the downside, antiviral vaccines are a challenging task because viruses mutate so rapidly. Therefore, the vaccines have to be updated regularly. In contrast, the antibacterial vaccines remain stable for a longer period. Compared to the vaccines, drugs are more broadly effective because they target a specific process, as in the case of Tamiflu and Relenza. 

Fig. 3.  Coronavirus (nature.com/articles/s41579-020-00468-6/figures/1)

Structure of
Coronavirus

Coronavirus is one of largest RNA viruses. It has a single strand of RNA that ranges 26,400 to 31,700 nucleotides in length. All proteins are coded by the genes on this strand. Similar to the flu virus, coronavirus has a membrane supported by structural proteins. Spike protein (S in Fig. 3) anchored in the envelope has the same function as hemagglutinin. It links with ACE2 receptors in the cells of the upper respiratory track to gain access into the cell. The virus also codes its own RNA polymerase that processes the viral RNA.

Fig. 4.  Coronavirus lifecycle (nature.com/articles/s41579-020-00468-6/figures/1, adapted)

Lifecycle of
Coronavirus

While there are a number of similarities between flu and coronaviruses, there is one important difference between the two. Their single-strand RNAs have opposite polarities. Coronavirus has +ssRNA and influenza has -ssRNA. The implication is that the coronavirus does not have to enter a cell's nucleus for replication. Getting in and out of the nucleus is a risky business. The +ssRNA viruses can start replicating as soon as they get inside the cell and exit quickly, before they are discovered. That reduces the risk of their destruction inside the cell. Coronavirus enters a cell when its spike protein engages with ACE2 on the cell surface (Step 1 in Fig. 4). The viral envelope is then cut open by proteases inside the cell (Step 2). A protease is an enzyme that breaks apart proteins. The viral RNA is replicated (Step 3) and new viruses are assembled from the replicated RNA (Step 4). They exit via the cell membrane (Step 6). Unlike the flu virus that grabs a piece of the cell membrane on its way out, coronavirus takes the membrane from an internal organelle of the cell called endoplasmic reticulum. Therefore, it is fully assembled before leaving the cell (Step 5).

Drugs and
Vaccines
Against
Coronavirus

The beige colored boxes in Figure 4 show some of the steps in the lifecycle of coronavirus that are suitable drug targets. The corresponding drugs are in brown boxes. For example, using protease inhibitors can prevent the proteases from cutting open the virus upon entry. Similarly, viral maturation inhibitors can prevent the progeny viruses from getting assembled.  Since the virus is fully mature when it is ready to leave the cell, there are no candidate targets for coronavirus that parallel the neuraminidase inhibitors for influenza. Unfortunately, there are no promising drugs in the pharmaceutical industry's pipeline against coronavirus. It is hard to kill the virus inside the cell without side effects. For example, protease inhibitors are a broad class of drugs that affect numerous biological processes in the body beside coronavirus. Therefore, a vaccine is the better option at this point. As we discussed in the influenza section, H and N proteins are the prominent proteins on the flu virus surface that our immune system can recognize and attack. Spike protein plays the same role in coronavirus. All vaccines currently under development or on the market target this protein. 

References
  • Dong, Y et al. (2020). A systematic review of SARS-CoV-2 vaccine candidates. Signal Transduction and Targeted Therapydoi.org/10.1038/s41392-020-00352-y.

  • Poduri, R et al. (2020). Drugs targeting various stages of the SARS-CoV-2 life cycle: Exploring promising drugs for the treatment of Covid-19. Cellular Signalingdoi.org/10.1016/j.cellsig.2020.109721

  • Samli, T. (2009). Influenza A: Understanding the Viral Life Cycle. Yale Journal of Biology and Medicine. 82(4). 153-159.

  • V'kovski, P et al. (2020). Coronavirus biology and replication: implications for SARS-CoV-2. Nature Review Biologydoi.org/10.1038/s41579-020-00468-6

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