The last major fragment meeting of 2019 took place at the Monash Institute of Pharmaceutical Sciences, Monash University, in marvelous Melbourne last week. This was the third Australian meeting devoted to fragments; you can read about the first, in 2012, here. With some 125 participants from four continents, two dozen talks, and nearly as many posters I’ll just try to capture major themes.
Biophysics played a starring role – if you haven’t already voted (right side of page) on which fragment-finding techniques you use please do so. Sarah Piper (Monash) discussed cryo-electron microscopy and showed some lovely high-resolution structures of proteins with bound ligands, though not yet with fragments. Sally-Ann Poulsen (Griffith University) described using native-state ESI mass spectrometry to discover new carbonic anhydrase binding fragments (see here). She uses a 96-well “nanoESI” chip to generate 5 µm droplets as opposed to the ~100 µm droplets typically fed into the instrument. Smaller droplets contain fewer molecules of salt and buffer, and thus generate cleaner spectra.
NMR screening is the go-to method for screening at Monash University, as highlighted by Martin Scanlon and multiple other speakers. Indeed, Monash has built their own version of Astex’s MiniFrag library – their MicroFrags include 92 compounds with 5-8 non-hydrogen atoms. Rebecca Whitehouse has screened these at 300 mM (yes, millimolar) by 15N-1H HSQC against the E. coli protein DsbA (EcDsbA) and found numerous hits, including at an internal cryptic site previously identified by Wesam Alwan (Monash). Encouragingly, the results were consistent with a crystallographic screen of the same library done at 1 M.
SPR was highlighted by Nilshad Salim (ForteBio) and in a separate Biacore user day, and is an essential tool for off-rate screening (ORS). ORS facilitates screening of crude, unpurified reaction mixtures, since the off-rate of a compound bound to a protein is not dependent on compound concentration (see here). Compound purification is a major time-sink, and avoiding it is a key component of REFiL, or Rapid Elaboration of Fragments into Leads.
As Bradley Doak (Monash) discussed, REFiL entails the parallel synthesis of compound libraries around a selected fragment in 96-well plates using diverse reagents and high-yielding chemistries such as amide bond formation, alkylation, and reductive amination. Reaction mixtures are evaporated, resuspended in DMSO, and screened using ORS; this has led to affinity improvements of ten-fold or better compared with the original fragment for four projects tested thus far.
Beatrice Chiew (Monash) presented a case study against the oncology target 53BP1. Screening 1198 fragments led, after catalog-mining and rescreening, to 25 hits, all quite weak. Applying REFiL improved affinities by up to 15-fold, with the best molecules around 10 µM. Beatrice noted that because SPR provides “on-chip purification,” active compounds could be identified even when the reaction yields were less than 10%. She did note that examining the raw data (sensorgrams in SPR-speak) is important to recognize and avoid false positives.
Similarly, Luke Adams (Monash) applied REFiL to the bromodomain BRD3-ET. After two cycles, he was able to improve a 230 µM fragment to a 1.5 µM binder. Importantly, the off-rates were similar for the purified molecules and the crude reaction mixtures.
And Mathew Bentley (Monash) is exploring the potential of REFiL using crystallography, or REFiLX. This led to a 60 µM binder against the notoriously difficult EcDsbA. That affinity is more impressive given that the previous structure-based design and synthesis of more than 100 compounds – aided by 25 crystal structures – had failed to break 250 µM.
Vernalis pioneered off-rate screening, and Alba Macias described the company’s latest developments in this area. In the case of tankyrase, a 700 µM fragment was used to generate 80 compounds, which took one chemist a couple days. This yielded a 350 nM binder, the structure of which bound to the enzyme was solved using the crude reaction mixture for soaking.
Following up on this success, Vernalis is exploring the limits of crude reaction mixtures for high-throughput crystallography. Although promising, Alba noted caveats for the two proteins tested. Unlike off-rates, crystallographic success is dependent on compound concentration, so low-yielding reactions can lead to false negatives. And as anyone who has spent time working with fragments can attest, a beautiful co-crystal structure is no guarantee of high affinity, so false positives (ie, no improvement in affinity over the starting fragment) can be a problem too.
Alba also gave a brief summary of the discovery of S64315/MIK665, a fragment-derived MCL-1 inhibitor discovered by Vernalis, Servier, and Novartis that is currently in phase 1.
MCL-1 is a member of the BCL-2 of family proteins, and BCL-2 itself is targeted by the second fragment-derived drug to be approved. Guillaume Lessene (Walter & Eliza Hall Institute) spoke about both of these proteins, as well as BCL-xL. Long-time readers may remember this selective BCL-xL inhibitor, discovered using second-site NMR screening. Blocking this protein leads to platelet cell death, but AbbVie researchers are ingeniously side-stepping this liability by conjugating a related small molecule to an antibody to reduce systemic exposure. The resulting ABV-155 may be the first antibody drug conjugate derived from fragments, and was said to be in phase 1.
There was quite a bit more, though in the interest of time (and readers’ patience!) I’ll stop here. But I must note before closing that this meeting launched the Australian Research Council-funded Centre for Fragment-Based Design. This is in some ways an Antipodean version of FragNet, though with a longer (five-year) funding period and the opportunity to include a few postdocs as well as graduate students. If you’re interested, please contact them.