Success in drug discovery is not necessarily fast or inevitable

The biotech industry rightly prizes speed: every day people die of diseases we are trying to prevent or cure. And developments can indeed happen quickly. Just eight years elapsed from the demonstration that a mutant form of KRAS was druggable to the approval of sotorasib, with less than three of those years spent in the clinic. Even more dramatically, it took less than a year from the first reports of SARS-CoV-2 to develop effective vaccines. But as two recent pieces in Nature Rev. Drug Disc. demonstrate, such speed is not necessarily the norm.

 

The first, by Asher Mullard, is entitled “FDA approves 100^th monoclonal antibody product.” This is a nice review of a remarkably successful therapeutic approach. But this triumph was not a foregone conclusion. Mullard traces the field’s origin to the mid-1970s, and while the first drug was approved in 1986, it took another eight years for the second. The article includes a timeline showing approvals by year, and it is interesting to compare this with FBDD-derived drug approvals since the 1996 publication of the seminal SAR by NMR paper. In the chart below, the first year on the x-axis is for antibody drugs; the second is for FBDD-derived drugs.

 

 

A quarter century after work began, new antibody approvals were still uncommon; Mullard notes that “antibody approvals have only been an annual event since 2006.”

 

Antibody-drug conjugates (ADCs) are an interesting subset that – as their name suggests – comprise an antibody linked to a small molecule, usually a toxin intended to kill cancer cells. Ten of these have been approved in the US, but while the first (gemtuzumab ozogamicin) was approved in 2000 most of the rest are recent, with six of them coming since the beginning of 2019.

 

By these standards the fact that only five fragment-derived drugs have been approved thus far isn’t surprising. Indeed, antibodies have some advantages: “whereas medicinal chemists can toil for years to find small molecules with activity against a given target, antibody discovery can take a matter of months.” Moreover, as the article continues, success in the clinic is roughly double that of small molecules.

 

The second article is by Christopher Austin, until recently Director of the National Center for Advancing Translational Sciences at the US National Institutes of Health. Titled “Translational Misconceptions,” it briefly enumerates and debunks false beliefs about translating new discoveries into drugs, which include:

 

- Translation does not exist 

 

- Translation is a “thermodynamically favored” process 

 

- Translation is straightforward and does not qualify as science 

 

- Translation is a unidirectional process 

 

- Once an investigational therapy gets into humans for the first time, regulatory approval

  and marketing are all but assured 

 

- Regulatory approval is the end of the translational process

 

Those of us in industry would probably dismiss these statements as naïve, but such perceptions are widespread. Indeed, Austin himself acknowledges that he “once believed unquestioningly in all of them.”

 

Each of these misconceptions invites discussion. To take just the last, the first approved ADC was pulled from the US market in 2010 when confirmatory trials showed that patients on the drug actually did worse than those on placebo. It was reapproved in 2017 after a better dosing schedule was established. In other words, it took 17 years after initial approval to figure out how to effectively use gemtuzumab ozogamicin, and 26 years from the beginning of the project.

 

Returning to the two successes mentioned at the top of this post reveals that their apparent rapidity does not tell the full story. The Tethering technology that eventually led to sotorasib was initially published more than twenty years ago, and researchers first used mRNA packaged in liposomes to transfect cells way back in 1989.

 

Amidst rapid visible progress it is easy to lose sight of the fact that much research goes nowhere very slowly. Even when successful, it might take decades to help patients. As Austin concludes, “only by advancing our common understanding of the complexity of translation, translational research and translational science will translational gaps be narrowed and eventually eliminated.”

Chemical couplets inhibit the GAS41 YEATS domain

Practical Fragments has covered bromodomains extensively, most recently just a couple months ago. But these epigenetic readers are not the only proteins that recognize acetylated lysine residues. Two years ago we highlighted fragment hits against one of the four YEATS domain proteins. A paper recently published in Cell Chem. Biol. by Jolanta Grembecka, Tomasz Cierpicki, and colleagues at University of Michigan tackles another member of the family.

 

The researchers were interested in the protein GAS41, which is amplified in multiple forms of cancer. The YEATS domain within this protein binds to acylated lysine residues in histone H3 proteins. However, unlike the deep pockets found in bromodomains, the acyl-lysine binding site in the YEATS domain consists of a partially solvent-exposed channel, making it a more challenging site to drug.

 

Nonetheless, an NMR-based screen of fragments (in pools of 10, each at 500 µM) led to compound 1, which produced multiple chemical shifts in a ¹H-¹⁵N HSQC experiment. Two different competition assays, fluorescence polarization (FP) and AlphaScreen formats, confirmed that compound 1 could compete with H3-derived peptides. Fragment growing led to compounds 7 and 11. (All IC₅₀ values below are from the FP assay; these are somewhat weaker than those from the AlphaScreen, but they more closely track binding affinities determined by isothermal titration calorimetry.)

 

A crystal structure of a compound closely related to compound 11 revealed that the molecule nearly fills the small binding channel, suggesting that further gains in affinity would be difficult. Indeed, no GAS41 inhibitors have been previously reported. However, the protein is dimeric, so the researchers decided to dimerize their molecule to bind to two YEATS domains simultaneously. This led to nanomolar molecules such as compound 19.

 

Not only was compound 19 potent in biochemical assays, it also disrupted binding of GAS41 to acetylated histone proteins in cells. Moreover, the compound inhibited growth of cancer cell lines with amplified GAS41.

 

This is a nice case study in fragment dimerization, an uncommon but interesting approach. The linking in this case led to a 44-fold improvement in affinity, which though impressive is far from synergistic, and is associated with a considerable loss in ligand efficiency. And although the micromolar potency of compound 19 in cells needs to be improved to generate a chemical probe, let alone a drug lead, these results nonetheless support the notion that targeting GAS41 could be a useful strategy for certain cancers.

Fragments in the clinic: TAK-020

The fifth fragment-based drug approved, sotorasib, acts by irreversibly binding to its protein target. However, despite increasing interest in covalent inhibitors, they are still rare among fragment-derived clinical candidates. A paper just published in J. Med. Chem. by Mark Sabat and colleagues from Takeda describes a new addition.

 

The researchers were interested in inhibitors of BTK, a kinase implicated in cancer as well as arthritis. Indeed, the drug ibrutinib, a covalent BTK inhibitor, has been widely credited with driving recent interest in covalent molecules. Back in 2015 we highlighted work from Takeda describing a fragment screen and optimization to a low nanomolar non-covalent lead. In addition to that fragment, the screen produced triazolone-containing compounds 5 and 6. Crystallography revealed that the triazolone moiety interacts with the kinase hinge region through multiple hydrogen bonds. Importantly, triazolones had not previously been observed as hinge binders. Thus the researchers decided to pursue the chemical space opened up by this discovery.

 

Although compound 6 had higher lipophilicity and lower ligand efficiency than compound 5, it provided better vectors for growing into the binding pocket. Changing the phenyl ring of compound 6 to a pyridyl gave a very slight boost in potency; growing led to low micromolar compound 11. Expanding the pyridyl to an isoquinoline further improved the potency to mid-nanomolar compound 18, which is still fragment-sized and highly ligand efficient. Crystallography suggested how to append an acrylamide warhead to engage cysteine 481 (the target of ibrutinib). This produced TAK-020, which overlays nicely onto compound 6. 

 

The whole campaign was remarkably fast: just 6 months of synthetic effort from fragment to TAK-020.

 

In general, the longer an irreversible inhibitor is incubated with a protein, the more potent it will appear, so IC₅₀ values can vary depending on the assay. A more meaningful number is k_inact/K_i, which turned out to be 205,000,000 M^-1s^-1 for TAK-020, more than 10-fold higher than ibrutinib. The compound demonstrated efficacy in a rat model of collagen-induced arthritis and behaved well in various safety assessments including two-week rat and dog toxicology studies.

 

Based on these results, the compound entered the clinic; the results of a phase 1 trial are reported by Eric Fedyk and colleagues in Clin. Transl. Sci. The compound proved safe and well-tolerated. Although TAK-020 has a relatively short half-life, it led to sustained reduction of BTK activity. (Since irreversible inhibitors by definition remain bound to their targets, their efficacy can be high even if rapidly cleared from the bloodstream)

 

And there the story ends. ClinicalTrials.gov reports that the two phase 1 clinical trials ended more than four years ago, and TAK-020 does not appear on Takeda’s pipeline. Four BTK inhibitors are already approved, with multiple others at various stages of clinical development, so perhaps the molecule was deprioritized for competitive reasons. The fact that TAK-020 first entered the clinic in March of 2015 is a reminder that what you read in the literature often reflects work completed many years ago. Nonetheless, this is a lovely fragment-to-lead story that is well worth perusing.

How fragments become leads

Our most recent poll asked how often synthetic challenges had kept researchers from pursuing a particular fragment or had impeded a fragment-to-lead project. Around two-thirds replied sometimes or often. A new open-access paper in Chem. Sci. by Rachel Grainger, Rhian Holvey, and colleagues at Astex does a deep dive into fragment-to-lead chemistry, provides a powerful visual tool, and ends with something of a call to action.

 

The researchers take as their starting points 131 fragment-to-lead success stories published from 2015 to 2019 and collated in a series of five J. Med. Chem. Perspectives. All of these started with fragments (< 300 Da) for which affinity increased by at least 100-fold, with the resulting leads having affinities of 2 µM or better. As the new paper points out, this could introduce “survivorship bias,” in that less successful projects are not included. However, as the point of the paper is to figure out what works, this likely strengthens the conclusions.

 

The targets themselves are fairly diverse: 24% kinases, 9% proteases, 36% other enzymes, 11% bromodomains, 14% other protein-protein interactions, and 6% other types of targets. The researchers closely examined how the leads related to the initial fragments. Full details are provided in the Supplementary Material (pdf). The researchers have also constructed a handy interactive viewer you can use to do your own analyses. Here is an overlay of an initial fragment (taupe space-filling) with the final lead (yellow surface).

 

What are the results? The first observation is that 93% of leads have at least one polar interaction (such as a hydrogen bond) that is conserved from the initial fragment. The most common functional groups making direct contacts to proteins are N-H hydrogen-bond donors (35%) followed by aromatic nitrogen hydrogen-bond acceptors (23%) and carbonyl oxygen hydrogen-bond acceptors (22%).

 

The second observation is that over 80% of fragments are grown from one or two vectors (examples of one and two, with the second the subject of the figure above). This is perhaps not surprising; growing from three or more vectors would likely result in portly molecules that may be more difficult to advance, venetoclax notwithstanding.

 

But the really interesting observation is that the majority of growth vectors (~80%) originate from carbon atoms. Moreover, more than half of the bonds formed are carbon-carbon bonds. For the non-chemists in the audience, this is significant because carbon-carbon bond forming reactions are not always straightforward, particularly in the presence of polar moieties.

 

In the early days of FBLD, one hope was that including functional groups such as amides in a fragment collection would facilitate fragment growing. The new paper suggests that this is naïve: a functional group in a fragment is likely to interact with the protein and so block potential growth vectors. Indeed, only 18% of growth vectors come from N-H groups, despite the fact that these are among the most synthetically accessible.

 

These findings thus explain why fragment-to-lead efforts can be so challenging. The researchers provide an example of a chemical series they ultimately abandoned due to poor synthetic tractability.

 

The paper also builds on earlier papers from Astex exhorting chemists to further advance chemical methodology. As they conclude:

An “ideal synthesis” of a lead would allow: (1) site-selective formation of bonds at all growing points of a fragment, (2) whilst being mild enough to be compatible with essential polar functionality, and (3) proceeding with minimal or no need for protecting groups….

 

We believe that further development of C-H functionalisation that is tolerant to polar fragments has the potential to transform FBDD.

If you’re in academia, this looks like a good opening for a grant proposal!