Although fragment-based screening has recently emerged as a synergistic approach to conventional in vitro or cell-based screens, there are a number of hurdles that must be overcome for the successful use of fragment-based discovery, advised Alex Kiselyov, president of deCODE Chemistry. “First, you need to know how robust your assay is. Incorporating multiple diverse checkpoints would help in assessing how accurate it is and how much you can trust your data. Running alternative assays is always a good idea as these provide for the independent assessment of data quality.”
A well-defined flow for developing drug candidates already has been established within the industry, noted Dr. Kiselyov. “At the earlier stages, one needs to make sure that the identified actives are biochemically sound and specific. As a next step, functional activity of hits or their optimized analogues is assessed in cell-based systems followed by proof-of-concept in vivo efficacy studies.
“In addition, a multitude of parameters need to be balanced including pharmacokinetics, metabolic stability, toxicity, and others. Many groups in the industry, including ours, have consistently found that a smaller lead-like hit provides a better starting point for a medicinal chemistry effort.”
deCODE is focusing on fragment-based protein crystallography. “We find that this is a highly effective approach to rapidly identify both novel ligands and alternative binding modes,” Dr. Kiselyov continues. “This approach, for example, allows us to work on difficult targets, including allosteric modulators and protein-protein interactions.
“The key to finding promising actives is our in-house Fragments of Life™ (FOL) library. This selection is compact (~1,300 molecules) yet chemically diverse. It contains molecules found in the cellular environment, their metabolites, as well as compounds that mimic protein architecture. In a sense, these are ‘molecular rulers’ for assessing binding areas within a target.
As the field progresses, new applications for fragment-based drug discovery are emerging. Andreas Kuglstatter, Ph.D., head of protein crystallography, Roche, reported, “Once you have found fragments that bind to your target protein, there are many things to do beyond growing or merging them to increase potency. Screening a large compound library for molecules similar to the identified fragments allows you to rapidly discover better molecules.
“For example, we were able to improve the activity of a kinase inhibitor by 100-fold in less than three weeks. You can’t do that if you have to synthesize new compounds. Another application is scaffold hopping, in which you replace the core of a known inhibitor with a fragment in order to develop a lead with different properties.”
Dr. Kuglstatter said that finding lead compounds with novel intellectual property (IP) can be challenging.
“High-throughput screening can find many potent hits, but the problem is IP space. Fragment screening is useful here, as well. We identified fragments with very distinct kinase binding motifs not described in the literature. We then made a custom library for each and tested their potency in a panel of 400 human kinases. So we now have highly attractive, off-the-shelf hits for many human kinases to jump start new projects.”
Fragments can also be used to explore protein flexibility and to enable rational selectivity design, noted Dr. Kuglstatter.
“One of the biggest issues with kinase inhibitors is selectivity. By cocrystallizing fragments with their targets, we identified unique protein conformations. This opened the door for the exciting application of selectivity design. Fragments can be optimized using structure-based approaches to bind stronger to your drug target and weaker to all other kinases. This translates into better in vivo safety. We have used these new applications for all small molecule programs where the target is a soluble protein.”
Protein kinases play key roles in intracellular signal transduction and, as such, are involved in a variety of disease states including oncology, metabolic, and immunological disorders. Because these targets are druggable, kinases have moved into focus as promising therapeutic drug targets. Proteros Fragments is targeting kinases among other relevant proteins with their fragment-based lead generation technology that employs cocrystallization of target and leads followed by structure-based optimization.
According to Gerhard Mueller, Ph.D., CSO and managing director, “In the area of kinases, we apply a conceptually novel retro-design strategy for which we have synthesized tailor-made fragments and specifically developed an assay technology for efficient fragment screening. The ability to engineer specific binding kinetic characteristics is the main advantage of this kinase inhibitor design approach. In addition, we intentionally avoid at the outset of a lead-finding program placing these seed fragments in the adenine-binding region, which usually translates into an improved IP position for the subsequent optimization campaign.”
Dr. Mueller says that most protein kinases in solution adopt a wide variety of different conformations, so inhibitors can be designed that show longer residence times on the target. “Different target conformations offer novel binding sites with unique topologies. Our strategy and fragment design concept that we pursue in-house is based heavily upon protein crystallography-derived structure information of the highest possible resolution. This provides a clear-cut assessment if the fragment of interest is indeed targeting the desired conformational state.
“The key element of the retro-design strategy is that distinct binding attributes can be constructed on the fragment state to create kinase inhibitors with slow dissociation rates. This translates into candidates with increased cellular and in vivo efficacy.”