Fragment-to-Lead
Medicinal Chemistry Publications
in 2020
Fragment-based
drug discovery (FBDD) has become a powerful approach
for interrogating drug targets and discovering new biologically active
compounds. Its success relies on screening
compounds of low molecular weight and low complexity (i.e., “fragments”)
during the hit-finding phase, employing sensitive and robust detection
methods. By using rational approaches such as structure-based drug
design, the hit fragments are efficiently grown, linked or merged
into larger lead compounds that can be progressed into preclinical
and clinical research. At present, six fragment-derived drugs have
been approved for clinical use, with Lumakras (sotorasib) being approved
in May 2021 and Scemblix (asciminib) being approved in October 2021.
Many more candidates that have been developed using FBDD are now undergoing
clinical studies. , Due to the reduced molecular
complexity of the initial chemical
matter, FBDD is an ideal breeding ground for the continuous development
of new technologies and their application in medicinal chemistry and
chemical biology. All this makes it valuable to monitor the FBDD literature.
Starting with FBDD literature published in 2015, we have been providing
the community with a tabulated overview of successful Fragment-to-Lead
(F2L) campaigns, and with commentary on trends extracted from the
scientific publications that were published in each calendar year. − Here we provide a similar review of FBDD articles published in 2020. Articles of interest were identified as described in an earlier
Perspective. In short, four different
strategies were followed: (i) A literature search using SciFinder and a variety of FBDD-related keywords; (ii) Articles that refer to a hallmark FBDD review; (iii) Careful scrutiny of
the 2020 issues
of Journal of Medicinal Chemistry, ACS Medicinal Chemistry
Letters, Bioorganic & Medicinal Chemistry , and Bioorganic & Medicinal Chemistry Letters , which in an
earlier analysis were found to have published many of the relevant
F2L studies; (iv) Publications that were discussed
on the well-known Practical Fragments blog. The literature collected from these
sources was evaluated and discussed.
As well as identifying remarkable FBDD developments and trends, we
used the criteria that have been applied since the first installment
in 2015 to select publications that qualify for inclusion in the table
of fragment-to-lead case studies. In brief: A fragment hit has a molecular weight (MW) < 300
Da. The fragment hit is identified using
a screening technology
(a biophysical, biochemical, or computational approach), adopted from
the literature or obtained by the deconstruction of a known ligand. The potency or affinity of the lead is equal
to or better
than 2 μM. The improvement in potency
or affinity from fragment
to lead is at least 100-fold. The publication
date (as defined by the primary citation)
for the article is in the calendar year 2020. It was noted in an earlier F2L perspective that more recent literature searches require careful analysis,
as an increasing number of relevant FBDD publications do not explicitly
use the term fragment in the title or abstract. This
was considered as a sign that FBDD was maturing. Conversely, we have
noted for some time that a growing number of publications have adopted
the term fragment or fragment-based without meeting our definition of FBDD. These studies use the term
“fragment” as a synonym for “substructure”,
for example, when combining features from different series of drug-like
molecules—an approach we appreciate, but refer to as “knowledge-based
design”. Authors, referees, and editors are encouraged to preserve
the designation “fragment-based” for those approaches
that actually determine (and ideally measure) the binding of the small
fragments as unique molecular entities. 

Careful
triage of the 2020 literature resulted in the identification
of 21 F2L studies that qualify for inclusion in . As always, the strict criteria for table
inclusion resulted in several notable studies that have not been tabulated.
In our discussion below, we outline some of these studies, which represent
new developments, trends, and excitement in the field. Analysis of the F2L entries in showed that 28% of the entries are kinase
inhibitors, a higher percentage than last year but close to the average
for the last 6 years . It is remarkable that the majority of the 2020 kinase F2L
programs did not rely on crystallography ( , entries 1, 2, 3, and 6). Apparently, the
structural understanding and molecular modeling capabilities suffice
to guide fragment optimization. In line with this, we note that all
2020 kinase F2L entries target the important and highly conserved
hinge region. , As the work on Protein Kinase
C ζ (entries 2 and 3) shows, fragment growing and fragment merging
(i.e., combining the decoration of different hit fragments that have
the same core) have proved to be very efficient. As seen in previous
years, most hit fragments are relatively “flat” (or
“2D-shaped”). Takeda’s work on ALK kinase (entry
4) and Leo Pharma’s work on JAK1 (entry 5) are good examples
of F2L programs that identify 2D-shaped hit fragments and incorporate
3D-shape including chirality during the lead-optimization programs. also shows
that proteases (5% of the table entries) and other enzymes (38%) remain
prominent targets in F2L studies. The work on Mycobacterium
tuberculosis InhA (entry 8) involves the replacement of a
carboxylic acid of the fragment hit by a sulfonamide. entries 9 and 10 both result
from a publication describing a program on the bacterial zinc metalloenzyme
LpxC. In this work, two different hit fragments were optimized into
unique lead compounds using structure-based design. Not captured in
the F2L table is a 2020 publication that describes FBDD approaches
to the development of enzyme activators, in this case targeting the
fungal glycoside hydrolase TrBgl2 as an enzyme with industrial applications. In the Perspective covering the 2019 literature, we also discussed an example of an enzyme activator.
Neither example meets the 100-fold improvement criterion specified
for inclusion in the F2L tables, but nevertheless they represent interesting
applications of FBDD. Protein–protein interactions (PPI)
also remain prominent.
This year had three F2L table entries for bromodomains (14%), two
of which reported crystal structures. The work by the Chinese Academy
of Sciences on BRD4 illustrates an efficient fragment-merging approach
in which a novel hit fragment is rapidly optimized by using substructure
features of a clinical candidate that had been developed by AbbVie
(who themselves used an FBDD approach to generate that clinical candidate,
previously reported as entry 22 in the 2017 F2L table ). Table entry 19 is for another class of PPIs, in this
case describing the development of inhibitors for the interaction
of the WDR5 scaffolding protein with the intrinsically disordered
transcription factor MYC, a promising cancer drug target. Here, too,
fragment merging has proved to be efficient. A 2020 study that just
missed the criteria for table inclusion focused on finding PPI inhibitors
for p47phox-p22phox (a complex that is key to activating NADP oxidase
isoform 2), work that led to a compound with a K i of 20 μM. A more technology-focused
study aimed to improve NMR fragment-screening protocols for finding
PPI inhibitors after the introduction of point mutations to weaken
the PPI, thereby making it easier for small screening fragments to
compete. Other studies that did not meet
the criteria for F2L table inclusion revealed a continued and growing
interest in PPI stabilizers, including the continuing prolific work
by Ottmann and co-workers on stabilizing interactions of the adaptor
protein 14-3-3. These recent studies included work showing that small
molecule stabilizers can differentiate between different PPI interfaces. Other publications by the same group describe
the use of fluorescence anisotropy as a readout for monitoring disulfide
tethering as an alternative to the previously
reported MS-based screening; and complementary
efforts to develop molecular glues using fragments that make a covalent
imine-bond with lysines of the target protein. These early results and the tools developed hold promise
for developing molecules that stabilize the interactions of 14-3-3
with various proteins. A spinout company has been established that
aims to further utilize these tools in drug development. The 2020 table also contains a GPCR example
(entry 20), a study
that delivered a negative allosteric modulator of GPR7. Careful fragment
library screening, hit exploration, and fragment growing resulted
in a lead without using structural information or biophysical screening,
i.e., types of data that remain difficult to obtain for GPCR targets. There are also several reports of FBDD
targeting RNA. − Although these programs have not yet produced leads
that fulfill
all F2L criteria for table inclusion, there is clearly growing interest
in identifying fragments that bind to RNA. The ongoing COVID-19
pandemic has underlined the importance of
scientific research to human health as evidenced by rapid development
of COVID vaccines. The FBDD community has also responded to this situation,
as illustrated by the very swift interrogation of the SARS-CoV-2 main
protease M Pro , although this work has not yet translated
into leads meeting our tabulation criteria for the year under review.
Only a few weeks after the COVID-19 pandemic struck, M Pro was identified as a promising drug target and interrogated with
covalent and noncovalent fragments using a combination of mass spectrometry
and high throughput X-ray analysis. Importantly,
as early as March 2020, nearly 100 structures of fragment-protein
complexes had been made public, providing valuable starting points
for inhibitors , thereby allowing the global research community to participate
in drug discovery. , These laudable efforts will hopefully
soon result in potent leads and a string of F2L publications that
originate from academia, industry and open-science initiatives around the world. A significant number
of the 2020 FBDD publications describe fragment
hit-finding efforts for microbial targets. entries 8–10 and 12 focus on specific
bacterial targets. All of these studies target (nonprotease) enzymes. entry 8 (targeting
the FAS II enzyme InhA that is involved in the reduction of long-chain
fatty acids) reports that the high activity of the lead compound does
not translate into antibiotic activity against Mycobacterium
tuberculosis . entries 9 and 10 target the
zinc metalloenzyme UDP-3- O -acyl- N -acetylglucosamine deacetylase (LpxC). While both series resulted in very potent leads, only the latter
resulted in in vivo efficacy. The work on DNA gyrase also resulted
in antibacterial activities, but significant differences were found
within the developed series of inhibitors, with efflux mechanisms
being the most likely cause for these differences, illustrating some
of the challenges in developing new antibiotics. While a string of other 2020 publications describe antimicrobial
FBLD hit-finding and exploration studies, they do not meet all the
criteria for F2L table inclusion. Examples include studies focused
on bacterial targets such as metallo-β-lactamase(MBL)NDM-1, dihydrofolate reductase from M. tuberculosis (MtDHFR), M. tuberculosis MabA (FabG1) and tRNA-modifying enzyme
TGT, where a hit fragment opens a transient subpocket that can be
exploited by a new series of ligands; tRNA modification enzyme TrmD, fungal
targets such as glucosamine 6-phosphate N -acetyltransferase
(Gna1), which is a key enzyme in the biosynthesis of an essential
fungal cell-wall component; parasite
targets such as the cysteine protease enzyme cruzain; allosteric binders for farnesyl pyrophosphate synthase
of Trypanosoma brucei ; bromodomain-containing factor 3 of Trypanosoma cruzi ; membrane-bound pyrophosphatases (mPPases);
virus targets (e.g., viral DNA-binding proteins Epstein–Barr
nuclear antigen 1 (EBNA1) and latency-associated nuclear antigen (LANA); and, as indicated above, several efforts to
probe and target SARS-CoV-2 proteins. Another interesting study developed
PqsR-targeting quorum-sensing inhibitors (a study that uses biophysical
screening and enthalpic efficiency evaluations, and introduces relatively
flexible linkers). presents
the screening technologies that were used to identify the hit fragments
in the F2L table entries. In 2020, we saw again that biochemical assays
were the most frequently used method for fragment-hit identification
(39%), followed by NMR screening (ligand-observed 17% and protein-observed
9%). The other well-established screening technologies are also represented
in the 2020 F2L table, including thermal shift assays (9%), which
were used in the successful M. tuberculosis InhA
program (entry 8) and BRD4 program (entry 18). For the first time,
the F2L table contains a publication (entry 12) that describes the
use of isothermal titration calorimetry (ITC) as the primary fragment-screening
method for DNA gyrase. As high protein consumption can limit screening
capacity, ITC has been used mainly as a secondary, orthogonal screen
to validate fragment hits. In this case, the use of ITC was enabled
by combining a focused screening library with an established protein
target. The use of ITC enabled the authors to select a fragment whose
binding to the target protein was enthalpy-driven and that was successfully
optimized into a lead compound. Although not reflected in the table, a prominent feature of the
collected 2020 literature involves the further development of fragment-screening
technologies. Several studies describe new and improved NMR screening
protocols, particularly those to increase throughput by automated
analyses of FBDD data, e.g., using freely available software tools
such as CcpNmr; or a new automated tool
called CSP Analyzer, which uses machine-learning-driven analysis to
assess multiple 2D HSQC spectra. 19 F NMR assays also continue to be improved, for example through
the use of novel broadband 19 F NMR, which allows faster
fragment screening and thus increases the throughput. Another study combines protein- and ligand-observed experiments
in a multiplexed screen that contains mixtures of fragments and bromodomain
proteins. Clearly, the NMR community
remains very active in providing increasingly efficient FBDD screening
technologies. The year 2020 also brought several reports of
fragment screening
using techniques that have not yet made it into our F2L tables. Among
others, two studies reported promising fragment screening using Weak
Affinity Chromatography (WAC) including for a GPCR target. , It will be interesting to see if these exploratory studies will
result in lead compounds. For X-ray crystallography, too, fragment-screening
throughput continues
to grow, as discussed in the context of SARS-CoV-2 above. In the form
of FragMAX, the XChem and Frag2Xtal platforms
now have a Swedish equivalent. Several reports also explore the use
of crude reaction mixtures in high-throughput X-ray crystallography. , These efforts aim to explore fragment hits more rapidly. As well
as X-ray crystallography, the 2020 literature also describes exciting
progress in applying cryo-EM to FBDD, , an emerging
technology that may open an expanded range of targets to structurally
enabled drug design. Considerable FBDD developments have also
continued in computer-aided
drug design (CADD) and virtual screening (VS). Among others, there
is a continuous effort to evaluate and improve docking algorithms
for the virtual screening of fragments, as their small size and low
complexity represent a real challenge, particularly for scoring. The
open source software SEED shows promising results and was successfully
used in fragment-library enrichment for a variety of targets. Another method for improving the ranking of
docked fragments (in this case using the rDock algorithm) is to evaluate
the docking poses generated in silico using a short dynamic undocking
experiment (a method dubbed DUck), essentially probing the robustness
of the hydrogen bonds that are involved in fragment-protein interactions. Several publications describe benchmarking
studies for the virtual
screening of fragment libraries. A useful data set (LEADS-FRAG) has
been released that allows proper comparison of various docking algorithms
and scoring functions. In our analysis
of the 2015–2020 F2L table entries that used virtual screening
to identify the hit fragments, we found that the docking algorithm
GLIDE was the most used . Unfortunately, GLIDE was not part of the benchmarking
study referred to above, and its VS performance
was therefore not compared to other algorithms. While there may be
a bias in that institutes with the resources to complete F2L studies
can also access the molecular-modeling tools provided by Schrödinger
(including GLIDE), we note that this particular software has successfully
delivered a F2L table entry every year since 2015. KinFragLib uses the structural kinome
database KLIFS and a computational fragmentation
method to split the cocrystallized ligands into fragments. The resulting
library consists of over 7000 fragments that are organized by subpocket
occupation. The data set is made available and can be used to explore
detailed binding characteristics and to generate novel inhibitors.
Other FBDD computational methods that have been developed help to
identify binding hot spots, again with
useful benchmarking sets being made available for testing and comparing
computational methods that identify binding hot spots (with emphasis
on FBDD). The potential of this approach was demonstrated by a group
who used in silico hot-spot identification to identify an unexplored
ligandable binding pocket outside the acetyl-lysine binding site of
a BRD4 bromodomain, prompting researchers to probe the site in question
with covalent binders (vide infra). New computational methods have been developed for fragment-linking
approaches, combining 3D structural information with machine-learning
techniques to design appropriate linkers. CADD tools like this may help fragment-linking strategies to achieve
their full potential: so-far, as a recent review article describes, this F2L strategy has had mixed success. Other
computational methods guide fragment growing, for example leading
to dual-activity epoxide hydrolase and LTA4 hydrolase inhibitors (table
entry 15), an approach that also involved machine learning using ligand-derived
and structure-derived fingerprints to distinguish actives from inactives.
Other in silico growing approaches that are described in the 2020
literature but did not meet the F2L criteria include a procedure called
FragPELE, which uses a Monte Carlo stochastic approach that allows
induced fit and the opening of cryptic pockets of the protein target. The continuous development of new CADD &
VS protocols confirms once again the prominence of the computational
chemistry community in FBDD. When
monitoring the molecular properties of fragment–lead
pairs, similar trends as discussed in the previous Perspective can
be identified. For the interested reader,
updated graphs that compare ClogP, LE, LLE, and deviation from planarity
of hits and leads are provided in the Supporting Information . For a detailed discussion of the different graphs,
see the 2019 F2L perspective. A noteworthy
observation for the 2020 data set is that multiple table entries obtained
100-fold activity improvement by carefully modifying and optimizing
the fragment scaffold (e.g., table entries 13, 14, 20). This is reflected
in that suggests
that the MW differences between the hits and corresponding leads of
the 2020 F2L studies are smaller than for the complete 2015–2020
data set. The complete fragment hit set from all F2L entries (2015–2020)
was analyzed using the Scaffold Classification Approach. In this analysis, a scaffold is defined by the
bonds that are part of a ring or that connect different rings (i.e.,
the acyclic side chains are pruned from the scaffold). Complexity
is defined by the size and shape of the scaffold, and is increased
by the number of rings, double bonds and heteroatoms. For example,
a phenyl ring is a less complex scaffold than a pyridyl ring (see ). Cyclicity is defined
as the ratio of the number of atoms that are part of the scaffold
to the number of atoms of the complete fragment. For cyclicity = 1,
there are no side chains; there are 17 hit fragments with cyclicity
= 1, including the most complex scaffold (the orange diamond symbol
in the top right corner of the plot, representing the mGluR5 hit described
as table entry 27 of the 2015 Perspective ). Cyclicity is lowered by more and larger side chains (see structures
in the column of diamond symbols with different hits containing the
phenyl scaffold on the left side of ). The analysis shows that the fragment hit
set is diverse in terms of complexity and cyclicity but a few prominent
scaffolds can be identified. Fifteen of all 152 F2L hits contain a
phenyl scaffold, and 8 hits contain a six-membered benzene ring fused
to a five-membered ring that contains 2 nitrogen atoms (see the benzimidazole
compounds in ). Strikingly, all the hit fragments contain at least one ring and
one sp 2 -hybridized carbon atom. The complete data set is
made available as part of a study that analyses the most common interactions
that hit fragments make and to identify the nominal synthetic growth
vectors. The theme of “3D fragment libraries” is also prominent, following the realization that unexplored scaffolds
represent a considerable opportunity. Especially when developing new chemistries, it makes sense to screen
the new scaffolds as fragments as this optimizes the chance of finding
hits. Publications from 2020 include DOS-derived and shape-diverse
fragment libraries that allow rapid derivatization of hits in different
directions; the design and synthesis
of shape-diverse 3D fragments; the synthesis
(and some screening) of a library of larger (seven-membered ring)
aliphatic heterocycles with 1,4-thiazepanones and 1,4-thiazepanes
as 3D-cores; a fluorinated Fsp 3 rich fragment library for 19 F NMR fragment screening; and a fragment library assembled from natural
products that contains a high fraction of sp 3 carbons (one
of the metrics used as a proxy for 3D shape). Another study focuses on improving the synthetic accessibility of
3D fragments by decorating sp 3 -carbon atoms in order to
grow from specific exit vectors. An unusual
approach to incorporating 3D shape is to develop metallo-fragments. It will be interesting to see how often this
type of fragment makes it into our F2L tables in the future; our 2019
Perspective included a table entry in which a rhodium conjugate was
incorporated during lead development. In 2020, as in previous years, there were also examples in
which
FBDD was blended with established or emerging orthogonal technologies. entry 16 shows work
on BRD4 that merged a fragment hit with a scaffold that was identified
using a DNA-encoded library. Other work on BRD4 (table entry 17) identified
a small fragment-like hit in a HTS screen that was successfully grown
into a lead-like compound using FBDD. Examples like these illustrate
that combining different technologies enables a plethora of drug discovery
opportunities. Another continuing theme is the identification
of novel binding
sites, such as that for tRNA-modifying enzyme TGT, where one fragment opens up a transient subpocket that
can be exploited by newly designed ligands. Other studies that did
not meet the lead criteria explore allosteric pockets in enzymes,
e.g., for farnesyl pyrophosphate synthase of the parasite T. brucei . The theme
of covalent FBDD continues to grow. Principles for the
design of electrophilic fragment libraries were reviewed by Keserű
et al., and a fragment hit that bound
covalently to its target has been successfully used in the F2L program
of Lp-PLA2 inhibitors (table entry 11). Also, as previously mentioned,
a combined mass spectrometry and X-ray approach was used to target
the SARS-CoV-2 main protease with electrophilic (and noncovalent)
fragments. Covalent-fragment screening
was also used to explore a ligandable binding pocket outside the acetyl-lysine
binding site of BRD4 bromodomain. The
use of cysteine-reactive fragments to target a computationally identified
hotspot allowed the exploration of a new binding site next to the
classic bromodomain binding site. Covalent
binders were long avoided in the pharmaceutical sciences, but this
reluctance has now eased. Some studies have even identified noncovalent
fragments and then introduced reactive electrophilic moieties to enable
a covalent link with the target. The work on cathepsin S inhibitors
(F2L table entry 7) is an example of this approach in which a noncovalent
fragment hit was identified using NMR screening and a nitrile warhead
was introduced to improve potency. A
similar 2020 example of adding a warhead during hit optimization is
the development of NSD1 tool compounds, although in this latter case
the potency change did not meet the criteria for table inclusion. Covalent binding is also used to probe proteins
with photoreactive fragments that cross-link with protein targets
(PhotoAffinity Bits or PhABits). Finally, two publications from 2020 described FBLD-derived clinical
candidates. Pfizer disclosed their ketohexokinase inhibitor PF-06835919, which entered phase 2 studies for nonalcoholic
fatty liver disease and type 2 diabetes mellitus. The fragment-to-lead
effort was entry 5 in our Perspective for 2017. And AbbVie described optimizing their pan-BET inhibitor
ABBV-075 to the BD2-selective compound ABBV-744; the initial H2L efforts that led to ABBV-075 was entry
22 in our Perspective for 2017. 

FBDD continues to play a major role in drug discovery, as demonstrated
by the progress reported in this Perspective. The impact is also apparent
when considering the fact that FBDD has resulted in a fifth (sotorasib)
and sixth (asciminib) approved drug, an
increasing number of compounds in clinical trials, and the impressive impact of interrogating COVID-19 targets. 

