What is a regulatory hotspot?
A regulatory hotspot is a privileged allosteric site used by nature to regulate the function of a protein in a precise manner. Nature often controls proteins through chemical modifications, called post-translational modifications (PTM). These modifications are diverse and include the addition of phosphate; carbon-containing groups such as methyl; and fatty chains such as myristolate. The modified protein can undergo a conformational change to interact with the regulatory hotspot or interact with another protein in a new way, leading to activation or inhibition of the protein. In select cases, small molecules can be designed to interact with the hotspot, thereby recapitulating the natural regulatory process.
What are the challenges associated with conventional approaches to drug discovery?
Conventional drug discovery approaches generally focus on the active site of the protein, the “engine” where fuel molecules such as adenosine triphosphate (ATP) are consumed or where chemistry is performed on the enzyme’s substrate. However, the active site presents several significant challenges:
Potency: Molecules targeting the active site must compete with very high concentrations of substrate or ATP. In the context of a cell or a living animal, this can result in a dramatic reduction in potency
Selectivity: Nature performs chemistry in only a select number of ways. Therefore, even if a molecule is able to successfully target an active site, it is likely that the molecule will undesirably modulate a series of other proteins, leading to potential side effects and poor tolerability in humans
Physical properties: Certain active sites are highly lipophilic (i.e. greasy) or charged, leading to drugs with similar properties. As a result, these molecules can be difficult to formulate into low dose, once daily pills that will result in better patient compliance.
How is HotSpot’s approach superior to traditional drug discovery?
Targeting regulatory hotspots provides a new and allosteric alternative for targeting proteins with an important role in disease. This confers several advantages compared with targeting the active site:
Potency: Molecules targeting a regulatory hotspot do not have to compete with high levels of substrate or ATP. As a result, hotspot inhibitors typically maintain potency in living systems.
Selectivity: Regulatory hotspots have evolved to be unique in order to exert precise control over the protein. As a result, molecules that bind to these sites are unlikely to affect other proteins.
Physical properties: Unlike active site inhibitors, molecules that target regulatory hotspots can exhibit attractive drug-like properties, offering the opportunity for low and less frequent dosing.
Novel pharmacology: Regulatory hotspots offer an entry point to drug targets that lack active sites (e.g., transcription factors, translation factors). We also observe differences in the pharmacology of hotspot inhibitors due to the potential to impact protein scaffolding.
How is targeting regulatory hotspots superior to other forms of allostery?
Not all allosteric sites are made equal. Some allosteric sites can have a dramatic effect on pharmacology, whereas others have a more limited effect. A regulatory hotspot is likely to be located within the part of the protein that controls function since Nature has honed this mechanism over billions of years to control the protein precisely. As a result, regulatory hotspots are more likely than other allosteric sites to drive important pharmacology.
Do all proteins have a regulatory hotspot? How many hotspots exist on one protein?
To date, HotSpot Therapeutics has identified a number of protein families that contain regulatory hotspots, within which druggable pockets have been uncovered. These include:
• E3 ligases
• Transcription factors
• Translation initiation factors
Through enhancements to our in silico and experimental platforms, we continue to expand the addressable target space amenable to our approach. For example, we are advancing lead programs targeting well-validated, sought-after targets that have proven difficult to address with existing drug discovery methods. To date, HotSpot has disclosed programs targeting protein kinases including:
S6 kinase (S6K): an immune-metabolic regulator of insulin sensitivity, lipid biosynthesis, and Th17 inflammation with potential to treat diseases such as NASH and Type 2 diabetes.
PKC-theta: a master regulator of immuno regulation with unique impact on both T-effector and T-regulatory cells with potential to treat inflammatory bowel disease (IBD) and other autoimmune disorders.
Are there examples of therapeutic agents that have been previously developed against regulatory hotspots ?
The metabolic enzyme Acetyl CoA Carboxylase (ACC) has been the source of industry interest for a few decades due to its role as the rate-limiting step in fat synthesis. Decreasing fat accumulation in the liver is an important step in treating diseases such as non-alcoholic steatohepatitis (NASH). Animal knockouts of ACC demonstrated improvements in both metabolic and liver health. Over 20 years, drug development efforts targeting ACC yielded non-selective molecules or molecules with poor drug-like properties.
While at Nimbus Therapeutics, we successfully drugged a regulatory hotspot on ACC. Nature regulates ACC via the phosphorylation of a flexible regulatory “tail” at one end of the protein. Once phosphorylated, the tail binds tightly into the interface between two copies of the protein i.e. a dimer interface. The binding of the tail prevents the protein dimerization, thereby inhibiting the protein. Small molecules were created that mimicked the effect of the phosphorylated tail. The ACC product portfolio was acquired by Gilead in 2016 and is currently in Phase 2b clinical studies for the treatment of NASH.
How does screening for traditional small molecules directed toward active sites vary from screening for small molecules targeting regulatory hotspots?
HotSpot Therapeutics has developed a proprietary computational and experimental platform called SpotFinderTM to uncover and drug regulatory hotspots across the proteome. This technology platform identifies regions on proteins that are putative hotspots by leveraging mechanism, structure and function data. To date, HotSpot Therapeutics has created an annotated pocketome consisting of more than 200,000 pockets derived from 130,000 structures.
Once identified, HotSpot then utilizes a range of experimental methods to validate the hotspots and applies a proprietary chemical library to create novel chemotypes and pharmacophores. This leads to attractive hit rates and drug-like chemical starting points.