Our latest fragment-based drug discovery paper against the p97 ATPase has been noticed and reviewed favorably by the widely-read Practical Fragments blog.
Here is an excerpt from that review:
“The protein p97 is important in regulating protein homeostasis, and thus a potential anti-cancer target. But this is no low-hanging fruit: the protein has three domains and assembles into a hexamer. Two domains, D1 and D2, are ATPases. The third (N) domain binds to other proteins in the cell. All the domains are dynamic and interdependent. Oh, and crystallography is tough. Previous efforts have identified inhibitors of the D2 domain, but not the others. Not to be put off by difficult challenges, a group of researchers at the University of California San Francisco (UCSF) led by Michelle Arkin and Mark Kelly have performed fragment screening against the D1 and N domains, and report their adventures in J. Biomol. Screen.
There is an excellent review paper from Dan Erlanson and Ben Davis that came out last year detailing some of the more common mistakes and artifacts that can arise in fragment-based screening campaigns (so-called “unknown knowns”). I encourage readers to go read the original paper. I have summarized some of the key points below:
1) Not checking compound identity to make sure what you think you purchased is what you actually have.
2) Low-level impurities in compound stocks can cause problems at the high concentrations used in fragment screens.
3) DMSO, commonly used to store fragments in plates, can act as a mild oxidant and is also hygroscopic.
4) Pan-assay interference compounds (PAINS) are common in many libraries and are found to give false positives to many targets.
5) Reactive functional groups in fragment hits can cause covalent binding or aggregation of the target.
6) Many fragments can show binding or inhibition while acting as aggregators rather than reversible binders. Including a small % of detergent can help eliminate these kinds of fragments from giving positive signals.
7) STD-NMR is very sensitive to weak binders, but because it relies on a relatively fast disassociation rate for the ligand, tight binders (<1 uM) can be missed by this method.
8) X-ray crystallographic structures are often taken as the “truth” when they are in fact a model of an electron density. Fragments can often be modeled into the density in incorrect orientations or in place of solvent atoms.
9) SPR methods are very sensitive to fragment binding, but can be confounded by non-specific binding of fragment to the target or chip, as well as compound-dependent aggregation.
10) Fragment hits should be validated by more than one method before embarking on optimization. They should also be screened for being aggregators by DLS or other methods.
Hepatitis C virus (HCV) is a single-stranded RNA virus that infects an estimated 180 million people worldwide.
In 2013, Gilead received FDA approval for a new HCV drug, Sovaldi (sofosbuvir), that inhibits viral replication by targeting the virus’s NS5B polymerase. Sovaldi has shown a very high cure rate (nearly 100% HCV suppression and sustained virological response) in clinical trials of previously untreated patients and has fewer side effects than pegylated-interferon and ribavirin therapies.
Sovaldi is a methyluridine-monophosphate prodrug: it is metabolized in the body back into methyluridine-triphosphate, which acts as a potent substrate mimic and inhibitor of the NS5B polymerase.
What is interesting about Sovaldi is the approach the scientists took to getting the inhibitor into the cell, relying on phosphoramidate prodrug technology that had been effectively used to develop anti-HIV drugs, but had never been applied before to this class of anti-HCV drugs.
During development, the researchers decided that they needed to deliver the charged methyluridine-monophosphate (rather than the neutral methyluridine) into the cell on the basis of two key observations: 1) the methyluridine triphosphate is the active compound against HCV NS5B polymerase, while the methyluridine alone is inactive (owing to very low conversion to monophosphate in vivo) and 2) the methyuridine monophosphated derivative can be anabolized in the cell back to the potent triphosphate form by an endogenous uridine-cytidine monophosphate kinase.
The phosphoramidate prodrug technology had never been applied to HCV inhibition until Solvadi.
The idea behind phosphoramidate prodrug technology is to create a membrane-soluble neutral prodrug derivative that can be metabolized in the liver by carboxylesterase-mediated cleavage and subsequent steps back to the monophosphate form.
The researchers applied the approach and after a significant amount of SAR investigation and PK/PD studies around the chemical composition of the phosphoramide substituents, they concluded that the structure of compound shown above was the optimal structure to deliver the methyluridine-monophosphate to the liver.
The result is a new generation of highly effective HCV therapeutics with few side effects that can make a significant difference in the lives of patients living with HCV.
Carmot Therapeutics, a small company located in San Francisco’s Mission Bay, has developed a very innovative drug discovery technology, called Chemotype Evolution (CE), that relies on fragment-based discovery but is different from traditional FBDD and HTS approaches in important ways.
The first important innovation is that CE relies on a “bait” molecule as a starting point for screening. The bait can be a known ligand, cofactor, or inhibitor. The bait is then derivatized with a linker moiety that allows it to become chemically bonded with every fragment in a proprietary library. This process generates a screening library that contains thousands of bait-fragment hybrids.
The most powerful aspect of CE is the ability to iterate over chemical space, allowing access to an exponential number of possible fragment-bait hybrids.
These hybrids are then screened against the target for binding using either biophysical or biochemical screening techniques in a high-throughput plate format.
The most powerful aspect of CE is the ability to iterate over chemical space, allowing access to an exponential number of possible fragment-bait hybrids. The method can be iterated with new “baits” derived from the best fragment hits of the previous round. Thus, instead of having 7,000 fragments in your library, after 3 iterations you access 7,000^3 possible combinations (343 billion possible compounds), selecting only the most target-relevant chemotypes at each stage.
The CE approach is similar in concept to the “tethering” approach pioneered at Sunesis, but differs in the fact that no protein engineering of cysteine residues needs to be performed. The bait molecule performs the role of the engineered cys, providing a “handle” that binds to the target and selects for complementary fragment binders.
Carmot Therapeutics just embarked upon their first major industry collaboration with the January 2014 announcement of a partnership with Amgen
Carmot Therapeutics just embarked upon their first major industry collaboration with the January 2014 announcement of a partnership with Amgen to use CE technology against two challenging targets. Identifying leads and developing hits will be carried out jointly between the companies, while clinical trials will proceed at Amgen. I think Carmot is definitely a company to watch given its innovative and potentially paradigm-shifting discovery technology and increasing interest from big pharma.