12 August 2019

Achieving maximum diversity with minimum size

One theoretical advantage of fragment-based drug discovery is the ability to efficiently explore chemical space: there are vastly fewer possible fragment-sized molecules than lead-sized molecules. That said, even fragment space is daunting; the number of possible molecules with up to 17 non-hydrogen atoms is about three orders of magnitude larger than the largest computational screen. Maximizing diversity is thus a key goal in designing fragment libraries, but how do you actually do this? A new open-access paper in Molecules by Yun Shi and Mark von Itzstein at Griffith University provides a practical new approach.

As the researchers point out, diversity itself can be a slippery concept. Functional diversity (ie, what targets are bound) is important but hard-won knowledge. Physicochemical diversity is by definition limited for fragments. That leaves structural diversity, as defined by “molecular fingerprints.” These can be as simple as the presence or absence of a fluorine atom, or can require complicated calculations involving, say, the distance between a hydrogen bond donor and acceptor in the lowest energy conformation of a molecule. In their paper the researchers focus on “extended-connectivity” fingerprints, which take into consideration the physical connectivity between different types of atoms.

But how can you actually quantify structural diversity? One possibility is by comparing molecules to see how different they are, as used for example in Tanimoto similarity assessments. Each additional molecule would be chosen to be least similar to those in a library. Alternatively, one could consider “richness,” how much of chemical space is covered, by calculating how many unique structural features (such as specific bond connectivities) are represented. Each additional molecule would be chosen to provide as many new molecular fingerprints as possible. Shi and von Itzstein propose a third approach, “true diversity,” that considers the number of unique features as well as their proportional abundances. In other words, a library with a higher true diversity would have a “more even distribution of proportional abundances.” The researchers note that this approach has been used in ecology for decades.

To see how their approach performs, the researchers started with a set of 227,787 commercially available fragments, all of which were roughly rule-of-3-compliant and scrubbed of undesirable functionalities. They also considered a subset of 47,708 fluorine-containing fragments. For both sets, they then assessed structural diversity as a function of increasing fragment library size using Tanimoto similarity, richness, and true diversity, as well as random sampling.

Naturally, as the size of a fragment library rose, the diversity increased. As expected, applying Tanimoto similarity or richness led to greater diversity at a smaller library size than did random sampling. This was even more true for true diversity. Interestingly, true diversity reached a maximum at 8.8% or 15.7% (for the full and fluorinated libraries) and then began to decline. This conceptually makes sense because commercial compounds themselves are unlikely to be truly diverse.

More importantly, just 1% or 2.5% of fragments were sufficient to achieve the same true diversity as the full sets. This corresponds to 2052 fragments for the complete commercial set, the structures of which are provided in the supplementary material. As the researchers note, this is comparable to the size of many commonly used fragment libraries.

The method is computationally inexpensive (it runs on a desktop), and should be a useful tool for both building and curating fragment libraries, real and virtual. Of course, diversity is not everything, and it probably makes sense to include privileged pharmacophores even at the cost of lower diversity. But as Lord Kelvin said, “when you can measure what you are speaking about, and express it in numbers, you know something about it.” This paper provides a quantitative approach for measuring diversity.

05 August 2019

Fragments vs RAS family proteins: A chemical probe

RAS family proteins are considered a holy grail of oncology research. Way back in 2012 we discussed a couple papers disclosing low affinity fragments that bind in a small, shallow, polar pocket found in KRAS, NRAS, and HRAS. At the time we wondered “whether this is a ligandable site on the protein.” Last year we highlighted a paper proving that the site is, in fact, ligandable, as exemplified by the mid-nanomolar molecule Abd-7. A paper just published in Proc. Nat. Acad. Sci. USA by Darryl McConnell and collaborators from Boehringer Ingelheim and Vanderbilt University (including Steve Fesik, who published one of the 2012 reports) describes successful development of another ligand. (See here for a fun animated description set to music.)

Consistent with the “undruggable” reputation of RAS family proteins, a high-throughput screen of 1.7 million compounds failed to find anything useful. In contrast, a library of just 1800 fragments screened using STD NMR and MST identified 16 fragments that bind to an oncogenic mutant form of KRAS, as confirmed by 2-dimensional (HSQC) NMR. A separate HSQC NMR screen of 13,800 fragments identified several dozen more, though all the fragments from both screens have dissociation constants weaker than 1 mM. SAR by catalog led to amine-substituted indoles such as compound 11, which modeling suggested could form a salt bridge to an aspartic acid side chain.


The pocket in which all of these molecules bind, between the so-called switch I and switch II regions of KRAS, is much smaller than typical drug-binding sites, but modeling suggested that fragment growing could pick up an additional hydrogen bond, leading to compound 15. Crystallography confirmed the predicted binding mode of this molecule, and informed additional structure-based design, leading first to compound 18 and ultimately to BI-2852, with low or sub-micromolar affinity for wild-type and mutant KRAS, NRAS, and HRAS as assessed by ITC. The researchers also confirmed that the enantiomer is about 10-fold less potent, thereby providing a control compound. Commendably, the researchers have made BI-2852 and the enantiomer available (for free!) to the research community as a chemical probe.

A crystal structure of KRASG12D bound to BI-2852 (cyan) compared with Abd-7 (magenta) reveals how shallow the pocket is; both molecules are largely surface-exposed. The conformational flexibility of the protein is also interesting: Abd-7 would not be accommodated by the protein conformation bound by BI-2852.

The biology is also quite interesting – and complicated. RAS family proteins behave as molecular switches, cycling between the “on” (GTP-bound) state and the “off” (GDP-bound) state, with these transitions assisted by other proteins. On-state RAS drives cell-proliferation and survival. Molecules that bind at the switch I/II pocket block the transition from off to on, but they also block the transition from on to off. Thus, cellular effects are modest. Moreover, BI-2852 hits all RAS isoforms, which could lead to unacceptable toxicity in animals.

This is a lovely paper, but I do quibble that the promise of the title – “drugging an undruggable pocket on KRAS” – remains to be demonstrated. First, both the biochemical and cell-based potency need to be further improved. As the molecule is already large, gaining this needed potency could come at the cost of physicochemical properties. Indeed, the researchers do not discuss the pharmacokinetics of BI-2852. And finally, as the authors themselves note, they will probably need to improve selectivity to spare one or more wild-type RAS isoforms.

What this work does establish indisputably is that the switch I/II pocket is ligandable, though not without effort, as indicated by the 42 authors. Whether or not the site is actually druggable may require another seven years to determine.