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.
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