Lesson 11 – Receptor Theory Adapted for RNA-targeted Drugs

Introduction

The theoretical foundation of the drug discovery industry, receptor theory, was laid in 1900 as scientists tried to explain the potency of natural poisons like curare. Interestingly, that theoretical construct led to the creation of thousands of new medicines, vast increases in knowledge and the building of the multi-billion dollar pharmaceutical industry was not proven until the first receptor was cloned in 1980 — 80 years after the industry began.

Prior to founding Ionis, in 1989 I adapted traditional receptor theory to address RNAs as “pharmacological receptors”. This was an essential step in creating the technology. As we have learned more, RNA receptor theory has evolved, but the basic theory has proven to be correct and enabled the treatment of ASOs (and siRNAs) in the context of pharmacology.

Why receptors?

If one thinks of drugs as bits of chemical information that must collide with other chemicals in our bodies to have the information in the drug molecule transferred to the body, it is clear that there are literally hundreds of trillions of chemicals with which a drug could collide in each of our bodies. If that is true (and it is), how do we explain the potency of some chemicals to cause profound changes, even death, when administered to a human being or animal? Scientists reasoned that chemicals that potently alter the behavior of a human, or other animal, must have specific targets to which they bind more tightly than all the other possible sites in the body, and those chemicals in the body must somehow amplify the effects of the exogenous chemical.

What are traditional drug targets?

Till the emergence of RNA targeted therapeutics, almost all drugs were designed to bind to specific proteins in the body. The proteins targeted were either “receptors” or enzymes.

A traditional receptor is a protein designed to bind to a specific chemical made by one type of cell that is secreted with the goal of altering the behavior of another type of cell. Epinephrine is a good example. It is made by one type of nerve cell, secreted by that cell, crosses a little space called a synapse and causes the recipient nerve cell to behave differently. So, a traditional receptor is a protein that has a binding site that very specifically interacts with its chemical stimulant (agonist) and is coupled to a cell signaling process that can amplify the effect of the natural agonist.

Enzymes are usually proteins that are designed to bind to a specific chemical made in the body (a substrate) and chemically alter the substrate, producing a new chemical, the product.

In either case, the drug target has a very specific function, interact with a very specific chemical made in the body and amplify the effects of the “agonist” or substate via activating a cellular pathway or creating a new chemical that can effect a significant change in the behavior of the responsive cell. Since there is a chemical made in the body for which the drug target is designed, most drugs are designed to bind to the same site as the natural chemical and either do what the natural chemical would do (agonists) or block the effects of the natural chemical (antagonist or inhibitor).

To sum up, traditional receptors are proteins expressed in one type of cell designed to bind and respond to a chemical made by another type of cell. They are directly coupled to a pathway that amplifies the effects of the natural agonist and cause profound changes in the behavior of the cell that is the recipient of the agonist. Consequently, the effects of an agonist on the recipient cell are quite rapid and most drugs are deigned to bind to the same site in the receptor as the natural agonist. Moreover, interactions that matter pharmacologically take place with receptor proteins that are mature and coupled to a pathway that induces a significant change in the behavior of the recipient cell.

RNAs as receptors for ASOs

Since we design ASOs to specific sequences in target RNAs, the cognate sequence in the RNA that binds an ASO can be considered a “receptor” for that ASO. The selectivity of the ASO for binding to the “receptor” sequence is directly defined by the number of nucleotide units that perfectly hybridize to the ASO, and we showed that an ASO 16 nucleotides or longer could precisely discriminate the desired binding sequence (receptor) from all other sequences in all the RNAs in a cell. So, ASOs can be rationally designed to bind to receptor sequences and by much more specificity than traditional drugs. Think of a button vs a zipper. A small molecule that binds to a protein receptor binds to a tiny spot in that protein and, thus, the drug tends to be not very specific and can fit into any button-hole. In contract, every zipper tooth (nucleotide) in an ASO receptor must fit perfectly.

Key differences between traditional receptors and RNA receptors

Number of potential receptor sites in proteins vs RNAs

For most proteins, there is a single site designed to bind to the natural agonist or substrate. Since most target RNAs are comprised of thousands of nucleotides and do need to bind to a specific sequence, in principle, an infinite number of 16-mer receptor sequences are available for RNase H1 ASOs. In fact, since we avoid RNA sequences, such as poly-pyrimidine or poly A sequences, that are repeated in many RNAs, the number of ASO receptor sites in a target RNA is fewer, but still vastly larger than the 1 or 2 sites that may be available in a protein receptor.

Design of ASOs is rational and rapid

Once we have the sequence of a target RNA, we design ASOs to bind to specific sequences in that RNA. Consequently, the initial design and testing of candidate ASOs is orders of magnitude more efficient and rapid than traditional small molecules.

ASO-RNA receptors are NOT directly coupled to a biological response

Irrespective of whether we design ASOs to cause degradation of the target RNA or simply alter the target RNA, the biological consequences of the ASO are NOT coupled directly to a biological response. So, ASO effects are typically slower developing than the effects of traditional drugs.

The number of molecules of a target RNA per cell is usually vastly smaller than the number of protein receptor molecules

Typical protein receptor molecules usually number >100,000 per cell. In contrast, for most target RNAs, the number of RNA molecules per cell is usually 1-10. This results in the fact that the number of target RNA molecules per cell makes no difference in ASO potency or effect, whereas the number of protein receptors per cell has a large effect on the activity of traditional drugs.

The maturity of protein receptors differs from RNA receptors

To matter, protein receptors must be fully mature and coupled to a biological response. In contrast, we target RNA receptors while the RNA is being processed and sent to sites in the cell where the RNA is used. This means that ASO technology can be more versatile, and the time required to cause a biological response may vary. For example, if we interact with a pre-m-RNA in the nucleus, a biological effect will not occur till the mature m-RNA in the cytoplasm is degraded.

Rates matter for ASO-RNA receptor sites

With ASOs, we are altering the lifecycle of a target RNA during its production, processing, and utilization by the cell. Thus, the rates of transcription, RNA splicing, and translation are critical factors affecting ASO activities. For most traditional drugs, rates do not matter.

RNAs have some sequences that are present in many or most RNAs

RNAs harbor sequences that are present in other RNAs that are used to direct processing or utilization of the RNA. Since ASO binding to such sequences would lead to effects on many RNAs, we must avoid these repeated sequences. Fortunately, these repetitive sequences are well understood and incorporated into ASO design algorithms.

Conclusions

Though there are important differences for RNA receptors vs protein receptors, most of these differences are well understood and the modified receptor theory that I devised in 1989 is an effective way to rationalize the drug effects of ASOs.

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