Small-molecule inhibitors that target protein-protein interactions in the RAD51 family of recombinases

Article abstract of DOI:10.1002/cmdc.201402428

The development of small molecules that inhibit protein-protein interactions continues to be a challenge in chemical biology and drug discovery. Herein we report the development of indole-based fragments that bind in a shallow surface pocket of a humanised surrogate of RAD51. RAD51 is an ATP-dependent recombinase that plays a key role in the repair of doublestrand DNA breaks. It both self-associates, forming filament structures with DNA, and interacts with the BRCA2 protein through a common "FxxA" tetrapeptide motif. We elaborated previously identified fragment hits that target the FxxA motif site and developed small-molecule inhibitors that are approximately 500-fold more potent than the initial fragments. The lead compounds were shown to compete with the BRCA2-derived Ac-FHTA-NH₂ peptide and the self-association peptide of RAD51, but they had no effect on ATP binding. This study is the first reported elaboration of small-molecular-weight fragments against this challenging target.

Full text of DOI:10.1002/cmdc.201402428

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DOI: 10.1002/cmdc.201402428
Small-Molecule Inhibitors That Target Protein–Protein
Interactions in the RAD51 Family of Recombinases
Duncan E. Scott,[b] Anthony G. Coyne,[b] Ashok Venkitaraman,[c] Tom L. Blundell,[a]
Chris Abell,[b] and Marko Hyvçnen*[a]
The development of small molecules that inhibit protein–pro-
tein interactions continues to be a challenge in chemical biol-
ogy and drug discovery. Herein we report the development of
indole-based fragments that bind in a shallow surface pocket
of a humanised surrogate of RAD51. RAD51 is an ATP-depen-
dent recombinase that plays a key role in the repair of double-
strand DNA breaks. It both self-associates, forming filament
structures with DNA, and interacts with the BRCA2 protein
through a common “FxxA” tetrapeptide motif. We elaborated
previously identified fragment hits that target the FxxA motif
site and developed small-molecule inhibitors that are approxi-
mately 500-fold more potent than the initial fragments. The
lead compounds were shown to compete with the BRCA2-de-
rived Ac-FHTA-NH2 peptide and the self-association peptide of
RAD51, but they had no effect on ATP binding. This study is
the first reported elaboration of small-molecular-weight frag-
ments against this challenging target.
Introduction
Protein–protein interactions (PPIs) are ubiquitous in nature and
play key roles in many cellular processes including apoptosis,
signal transduction, gene expression, and DNA repair and repli-
cation. It is not surprising that the inhibition of certain PPIs has
been hypothesised to have implications in many diseases, in-
cluding cancer.[1,2] PPIs offer many potential targets for small-
molecule intervention in disease, but are a challenging and un-
derrepresented target class in modern drug discovery. PPI sites
bury greater surface areas than protein–ligand binding sites,
and are generally featureless by comparison,[3] often lacking
major concave binding pockets that naturally bind small mole-
cules.[4] PPI inhibitors consequently tend to bind in many small-
er pockets.[5] As a consequence of these inherent differences
between PPI sites and traditional targets, PPI inhibitors tend to
be larger, more hydrophobic, and form fewer hydrogen bonds
than the small-molecule ligands of enzymes.[6]
Nutlin compounds, which bind MDM2 at the p53 binding
site.[7] However, HTS methodology has enjoyed limited success
against this target class.[8] For example, Fairbrother and co-
workers reported the failure of HTS in targeting the interaction
between the BIR3 domain of XIAP, and the partner protein
Smac; in this case, however, a screen of structure-based libra-
ries designed on the Smac binding epitope “AVPI” proved suc-
cessful.[8]
Fragment-based drug discovery (FBDD) has met with some
success in the field of PPI inhibitors. Briefly, in FBDD, low-mo-
lecular-weight compounds are screened at relatively high con-
centrations to identify weak, yet efficient binders.[9] Typically,
structural information regarding the fragment–protein complex
is then used to guide fragment “growth”—chemical elabora-
tion of the original hit—in order to establish additional interac-
tions. Different fragments may be found to bind simultaneous-
ly in adjacent pockets, in which case there may be an opportu-
nity for fragment “linking” to greatly improve potency, as was
reported by Petros et al. with a series of Bcl-2 inhibitors.[10] The
interaction between the anti-apoptotic Bcl-2 and BH3 proteins
is defined by a deep hydrophobic groove in Bcl-2 and has
been successfully targeted with compounds that have shown
antitumour activity in xenograft experiments,[11] and recently in
patients.[12] Further compounds have now begun to show
promise in clinical trials.[13,14] A fragment screen against the
bacterial membrane-anchored protein ZipA identified frag-
ments that bind to the C-terminal region of ZipA. Compounds
were then developed and were shown to block the binding of
an a-helix peptide from FtsZ.[15] Fesik and colleagues recently
used a fragment-based approach to develop small molecules
that prevent Sos binding to K-Ras by binding at a surface site
of K-Ras.[16] Despite these successes, identification of fragments
at PPI sites is not always possible. For example, fragmented
Despite these challenges, there have been a number of suc-
cesses in the development of PPI inhibitors, with various strat-
egies showing success against a diverse set of targets.[1] High-
throughput screening (HTS) is a common approach in modern
drug discovery, and was used successfully to develop the
[a] Prof. T. L. Blundell, Dr. M. Hyvçnen
Department of Biochemistry, University of Cambridge
80 Tennis Court Road, Old Addenbrooke’s Site, Cambridge, CB21GA (UK)
E-mail: mh256@cam.ac.uk
[b] Dr. D. E. Scott, Dr. A. G. Coyne, Prof. C. Abell
Department of Chemistry, University of Cambridge
Lensfield Road, Cambridge, CB21EW (UK)
[c] Prof. A. Venkitaraman
Medical Research Council Cancer Unit
University of Cambridge, Hills Road, Cambridge CB20XZ (UK)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402428: experimental procedures for
ITC, synthetic methods, and compound characterisation.
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MDM2 compounds, which were screened as fragments, were
not found to show detectable activity unless they filled two
sub-pockets and were at the higher end of the typical molecu-
lar weight of fragments.[17] Also, Van Molle et al. reported being
unable to detect the binding of fragments dissected from
high-potency compounds against von Hippel–Lindau pro-
tein.[18]
er pocket, across the central b-sheet of RAD51. As BRCA2 is in-
volved in localisation of RAD51 and formation of a nucleopro-
tein filament on DNA at sites of double-strand breaks, we hy-
pothesise that disrupting the interaction between RAD51 fila-
ment protomers and between RAD51 and BRCA2 is a valid
strategy to increase the susceptibility of tumour cells to radia-
tion and DNA-damaging agents.[22] Therefore, the development
of a small molecule that disrupts these interactions by binding
at the common FxxA binding site is of interest both as a chemi-
cal tool and as a potential therapeutic compound.
In this work we targeted the protein–protein interaction be-
tween RAD51 and the tumour suppressor protein, BRCA2.
RAD51 is a recombinase involved in the homology-directed
repair of DNA double-strand breaks, forming an active filament
with single-strand DNA in an ATP-hydrolysis-dependent pro-
cess.[19] Structurally, its interaction with BRCA2 is mediated by
eight BRC repeats that are characterised by a common FxxA
motif (Figure 1A).[20] In addition, the FxxA sequence motif
We previously published fragment-screening results using
a humanised RAD51 surrogate from Pyrococcus furiosus, which
we refer to as HumRADA2 (previously referred to as MAYSAM
RadA).[23] In this surrogate protein, the N-terminal domain with
the oligomerisation sequence was removed to monomerise
the protein and expose the FxxA binding site for
screening, and six surface residues were mutated
around the Phe pocket in order to humanise the pro-
tein in this region. The tetrapeptide Ac-FHTA-NH2, de-
rived from the BRC4 peptide, was found to have a KD
value of 250 mm, and the crystal structure in complex
with humanised RadA was solved (Figure 1D,E). A set
of fragments that bind at the PPI interface in the Phe
pocket of the FxxA motif were also identified, includ-
ing indazole (KD =1000 mm), l-tryptophan methyl
ester 1 (KD =570 mm, Figure 1F), and 4-methyl ester
indole (KD =1300 mm).
Results and Discussion
Herein we present the development of these frag-
ment hits from millimolar to low-micromolar potency.
Initially, small modifications were made to 1, which
led to only small changes in potency (Table 1 com-
pounds 2–4). Compounds with more significant, yet
still relatively modest modifications were then syn-
thesised and tested by isothermal titration calorime-
try (ITC) (Table 1). The benzyl ester 5b showed
Figure 1. A)–C) Cartoon representation of RAD51 binding sites. In addition to an ATP
binding site and a BRC repeat helix binding site (“Velcro” region), a surface binding
pocket (green) binds the FxxA epitope of the BRC repeats of BRCA2 (A, blue) and the
self-oligomerisation peptide of RAD51 (B, orange). The Ac-FHTA-NH2 tetrapeptide is indi-
cated for clarity (C, red). D) Crystal structure of Ac-FHTA-NH2 (red) bound to RAD51 (PDB
code 4B3B). E) Highlight showing surface pocket of RAD51; the contacting surface is rep-
resented in green. F) l-Methyl ester tryptophan 1 fragment previously identified that
binds in the Phe pocket of HumRADA2 (PDB code 4B2I).[21]
a small increase in potency, KD =200 mm, relative to
the parent fragment 1. Extending the carbon chain
by another atom gave strikingly better potency: com-
pound 5c, which at 35 mm, exhibited an approximate
10-fold improvement over the parent fragment 1. An
investigation of the length of the ester carbon chain
revealed that the ethyl linker is optimal; both the
occurs in the N-terminal linker region of RAD51, enabling it to
self-oligomerise through interaction with this region to form
RAD51 filaments (Figure 1B). The FxxA oligomerisation motif
of RAD51 is highly conserved across several species,[21] and six
of the eight BRC repeats conserve the same motif across sever-
al mammalian BRCA2 proteins.[20] The structural basis of this
FxxA motif was elucidated by Pellegrini et al., who solved the
X-ray crystal structure of truncated human RAD51 in complex
with the fourth BRC repeat (BRC4) from human BRCA2.[21] The
structure revealed FxxA as part of a b-hairpin across the sur-
face of RAD51, with Phe1524 of BRC4 in a surface pocket on
RAD51, and Ala1527 contacting the surface in a much shallow-
phenyl ester 5a and the phenpropyl ester 5d showed poorer
potency (Table 1). An SAR study focused around the phenyl
group of compound 5c identified substituents that had only
a moderate effect on potency and those that did not bind
(KD >200 mm). Changing the ester group to an amide led to
a large decrease in affinity (Table 1, compound 5e). A range of
substituents are tolerated on the phenyl ring, with methyl,
fluoro, and hydroxy groups (Table 1, compounds 5 f–j), and no
significant change in KD was observed. However if 4-bromo
and 4-methoxy groups were introduced to the phenyl ring,
there was a significant decrease in affinity, presumably due to
a steric clash with the protein at the para position (Table 1,
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compounds 5k and 5l). Replac-
ing the phenyl ring with cyclo-
hexyl or indole rings was also tol-
erated (Table 1, compounds 5m
and 5n).
Table 1. SAR for tryptophan ester series binding to HumRADA2 (ITC).
Further modification of com-
pound 5c also focused attention
on the amino group. Modifica-
tion with acetyl had no signifi-
cant effect on affinity (Table 1,
compound 6b). However, the in-
troduction of benzoyl onto the
amino group led to a significant
increase in affinity: KD =3 mm
(Table 1, compound 6a). A meta-
nitrophenyl substituent, a cyclo-
hexyl amide, and a phenethyl
amide all failed to give detecta-
ble activity (Table 1, compounds
6c–e).
Inspired by the indole scaffold
from tryptophan and other frag-
ments previously reported to
bind in this pocket,[23] we devel-
oped a series of sulfonamide de-
rivatives of tryptamine. We rea-
soned that the indole motif
would anchor in the Phe pocket,
and a sulfonamide linkage with
hydrogen bond acceptors and
donors would have the ability to
form hydrogen bonds to proxi-
mal side chains around the Phe
pocket such as Gln217 and the
backbone of Tyr202, to which
the FHTA peptide also forms hy-
drogen bonds. Initially the
phenyl sulfonamide 7a and the
benzyl sulfonamide 7b were syn-
thesised and tested by ITC. Inter-
estingly, only the phenyl sulfona-
mide 7a exhibited evidence of
Compd
R1
R2
LE[a]
0.28
LLE[b]
1.9
KD [mm][c]
1
H
H
570[23]
2
3
4
0.24
0.22
0.22
2.2
2.0
2.6
1500Æ24
810Æ19
890Æ15
5a
5b
5c
5d
H
H
H
H
0.24
0.23
0.26
0.22
0.7
190Æ5
200Æ8
35Æ4
0.7
1.1
0.02
160Æ7
5e
5 f
H
H
–
–
>200
25Æ5
0.26
1.1
5g
5h
H
H
0.26
0.26
1.5
0.7
27Æ12
30Æ9
5i
5j
5k
H
H
H
0.26
0.26
–
0.7
0.8
–
30Æ7
22Æ4
binding, with
a KD value of
>200
28 mm, indicating specific re-
quirement for a directly attached
phenyl ring. No activity was de-
tectable for the benzyl substitu-
ent 7b or for the methyl sulfona-
mide compound 7i. The SAR
around the phenyl ring was ex-
plored with compounds 7c–h,
and the results are listed in
Table 2. The introduction of a bro-
mine atom at the 4-position of
the phenyl ring led to a decrease
in affinity to 92 mm; however, if
the bromine substitution was at
5l
H
H
–
–
>200
28Æ9
5m
0.27
0.8
5n
6a
H
0.23
0.24
1.0
0.5
35Æ9
3Æ1
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were unsuccessful. Often poor
electron density for only a single
ring was observed in the Phe
pocket, but could not be unam-
biguously assigned to either
ring of the molecule. It is possi-
ble that there is some specific
molecular recognition in the
pocket, but disorganisation in
the binding modes as the mole-
cule emerges from the Phe
pocket, providing hydrophobic
interactions with the protein
surface. Therefore, in X-ray crys-
tallography we might see part
of a ligand bound in the Phe
pocket, but a superposition of
binding modes outside the
pocket would render this part of
the ligand invisible. Hence, the
location of the binding site of
compound 5c was investigated
by ITC experiments (Figure 2).
Table 1. (Continued)
Compd
R1
R2
LE[a]
0.25
LLE[b]
1.5
KD [mm][c]
18Æ1
6b
6c
6d
6e
–
–
–
–
–
–
>200
>200
>200
[a] Ligand efficiency (kcalmolÀ1 per heavy atom). [b] LLE=pKDÀclogP; clogP was calculated with Instant
JChem (ChemAxon). [c] Estimated errors in KD are given, generated by fitting a single-site binding model
(Origin software version 7.0552).
the 2-position, the affinity increased to 3.1 mm (Table 2, com-
pounds 7c and 7d). The inclusion of NO2 or NH2 groups at the
3-position had no significant effect on affinity (Table 2, com-
pounds 7e and 7 f). Interestingly, if 4-methoxy was included
on the phenyl ring, a decrease in affinity was observed, but if
this was replaced with the structurally similar benzodihydrofur-
an (Table 2, compounds 7g and 7h) the affinity was recovered
to 34 mm. Unsurprisingly, replacing the phenyl ring with a thio-
phene ring, which is sometimes used as a phenyl isostere,
gave a KD value similar to that of the phenyl group (Table 2,
compound 7j). We then explored SAR around substituted thio-
phenes: compounds 7k–p. Clear improvements in potency
were observed with benzothiophene (Table 2, compound 7p,
KD =1.6 mm) and halogenated thiophenes, most notably thio-
phene 7m, which has a KD value of 1.3 mm. We also explored
a selection of more polar five-membered heterocycles, a substi-
tuted isoxazole 7q, a furan 7r, and a methylimidazole 7s.
Whilst the furan possessed similar potency to the phenyl com-
pound 7a, the isoxazole showed a drop in potency to 110 mm.
The imidazole 7s was the amongst the weakest binding of all
compounds (KD =420 mm). Taken together, the SAR seems
largely driven by a hydrophobic interaction with the substitu-
ent on the sulfonamide; para substituents on phenyl rings are
not well tolerated relative to ortho substituents, suggesting
a steric clash or unfavourable interaction on the protein sur-
face in a manner similar to that observed for the tryptophan
ester series. Substituents on the thiophene scaffold yielded
a number of potent compounds; however, it appears that hy-
drophobic groups appear to give the largest increases in po-
tency. The more polar five-membered heterocycles showed de-
creased potency relative to the unsubstituted phenyl ring.
Unfortunately, attempts to crystallise and solve the struc-
tures of the more potent sulfonamide compounds and trypto-
phan ester compounds in complex with the target protein
Binding of 5c could be detected directly against the HumRA-
DA2 protein with a humanised FxxA binding site (KD =35 mm),
but not against the unhumanised wild-type RadA protein (Fig-
ure 2A). The importance of the humanising mutations around
the Phe pocket to binding is highly suggestive of 5c interact-
ing at this site. Competitive ITC experiments also implicated
the FxxA binding site (Figure 2B). Briefly, titrations of tetrapep-
tide Ac-FHTA-NH2 (KD =250 mm), the wild-type RadA oligomeri-
sation peptide (KD =7.0 mm), and ATP (KD =2.5 mm) were per-
formed individually against apo protein to measure their re-
spective KD values (top row titrations, Figure 2B). The KD mea-
surement of each ligand was then repeated, but in the pres-
ence of 200 mm 5c (bottom row titrations, Figure 2B). As
expected for an FxxA site binder in this system, the binding of
ATP was not affected by pre-incubation of the protein with 5c,
but 5c competes with and blocks the binding of both the self-
oligomerisation peptide and the Ac-FHTA-NH2 tetrapeptide.
Taken together, these experiments provide strong evidence
that the FHTA interaction site is the specific binding site of 5c.
Analogous competitive ITC experiments with 7m also demon-
strated blockage of the binding to humanised RAD51 of the
oligomerisation peptide but not ATP, consistent with specific
binding at the FHTA binding site (data not shown).
It has previously been observed with thrombin inhibitors
that substituting a terminal cyclopentyl ring for a cyclohexyl
group in the S3/S4 specificity pocket can have marked changes
on the binding thermodynamics and the electron density ob-
served in X-ray crystallography.[24] The binding of the cyclopen-
tyl compound was enthalpically driven, and the cyclopentyl
group was clearly observed bound to the protein by X-ray
crystallography. In contrast, the cyclohexyl compound, which
had the same KD value, was driven to bind by favourable en-
tropic factors; the cyclohexyl appendage was disordered and
could not be observed crystallographically. For this reason, and
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the lack of clear electron density for our compounds, we ana-
lysed the entropic and enthalpic contributions to our devel-
oped series in greater detail. Comparison of the binding ther-
modynamics of the original fragment 1 and the modified frag-
ments, compounds 2–4, is shown in Figure 3 (Table S1 in Sup-
porting Information). The binding of the fragments is enthalpi-
cally driven, overcoming an entropic penalty of binding of
~2 kcalmolÀ1. The binding of the oligomerisation peptide,
which binds with KD =7.0 mm, is almost entirely driven by fa-
vourable enthalpic contributions. However, for the representa-
tive lead series compounds 5c, 6a, and 7m, it is clear that in-
creased potency has largely been achieved by optimisation of
favourable entropic contributions. This switch in binding from
enthalpy-driven fragments to an entropy-driven series is no
doubt in part due to the hydrophobic nature of the attached
groups. The favourable entropic component of binding of the
lead series may reflect a disordered binding mode and may
explain the difficulties in obtaining a crystal structure in com-
plex with the protein. In retrospect, we can speculate that in-
troducing hydrogen bond acceptors and donors to both rings
might anchor both ends of the molecule, fixing a compound
into an ordered binding mode. Although not necessarily im-
parting potency, presumably such a molecule would be more
amenable to X-ray crystallography.
Table 2. SAR for substituted tryptamine series binding to HumRADA2
(ITC).
Compd
7a
R
LE[a]
0.30
–
LLE[b]
1.6
–
KD [mm][c]
28Æ6
7b
>200
7c
0.25
0.34
0.4
1.8
92Æ22
7d
3.1Æ0.1
7e
7 f
0.27
0.28
0.23
0.25
1.9
2.4
1.2
1.7
18Æ4
31Æ6
7g
7h
120Æ10
34Æ12
We have elaborated initial fragment hits that bind in a small
surface pocket of a humanised RAD51 construct and have in-
creased the potency by approximately 500-fold to ~1 mm.
Drug development is a multidimensional optimisation prob-
lem, however, and affinity for the target is not the only consid-
eration. The utility of ligand efficiency metrics in drug discov-
ery to assess the relative importance of affinity, logP, and mo-
lecular weight has recently been reviewed.[25] Ligand efficiency
(LE) is defined as the free energy of binding in kcalmolÀ1 per
non-hydrogen atom. An examination of the LEs of the com-
pounds in each series reveals some interesting differences. In
the tryptophan ester series, the starting fragment 1 (KD =
570 mm) has an LE of 0.28. This value is slightly below the
value of 0.3 kcalmolÀ1 per heavy atom, which is commonly ac-
cepted as a reasonable threshold for non-PPI targets; however,
it has previously been noted that inhibitors of PPIs tend to
have a lower LE of 0.24.[26] The developed phenethyl ester 5c
(KD =35 mm) had an LE of 0.26, which fell subsequently to 0.24
for 6a (KD =3 mm), the most potent compound in this series.
Conversely, several of the more potent compounds in the sul-
fonamide series possessed an improved LE, especially the
most potent compound 7m, which had an LE of 0.36. This is
a desirable achievement for any FBDD programme and even
more impressive for small molecules that bind at a PPI site.
Lipophilic ligand efficiency (LLE) is a ligand efficiency metric
that penalises molecules which are highly lipophilic and is de-
fined as the pIC50 (or pKD) minus clogP.[27] The higher the LLE
value, the more potency is generated for a given lipophilicity.
Although the potency of the compounds was not optimised
with LLE in mind, it is interesting to retrospectively examine
the LLEs. It has been suggested that optimised drug molecules
should aim to possess an LLE of 5–7, although absolute values
depend on the method used to calculate clogP. For the tryp-
7i
7j
–
–
>200
0.33
1.9
16Æ6
10Æ1
13Æ1
7k
7l
0.32
0.32
1.2
1.3
7m
7n
7o
0.36
0.33
0.27
1.5
0.6
1.3Æ0.1
2.3Æ0.4
67Æ29
À0.4
7p
7q
0.33
0.25
1.8
2.4
1.6Æ0.6
110Æ12
7r
7s
0.32
0.22
2.6
1.8
18Æ2
420Æ74
[a] Ligand efficiency (kcalmolÀ1 per heavy atom). [b] LLE=pKDÀclogP;
clogP was calculated with Instant JChem (ChemAxon). [c] Estimated errors
in KD are given, generated by fitting a single-site binding model (Origin
software version 7.0552).
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potency and actually increasing
LE, suggesting the sulfonamide
series is more attractive in terms
of their suitability toward devel-
opment.
Conclusions
Although we were unable to
obtain X-ray crystal structures
for our best compounds bound
to the protein, direct and com-
petitive ITC experiments strongly
implicate the FxxA binding
region as the most probable
binding site.
A
retrospective
analysis of the thermodynamics
of ligand binding reveals
a strong entropic component,
suggesting that the increase in
logP of the compounds caused
the potency to increase through
hydrophobic interactions with
the protein surface. With a lack
of hydrogen bonds that might
be thought to specifically orient
a molecule, we hypothesise that
a multitude of binding poses for
the lipophilic moieties of the
lead molecules may be superim-
posing in X-ray crystallography
and causing a disappearance of
ligand electron density. There-
fore, a future strategy for elabo-
ration of these compounds will
involve introducing a balance of
lipophilicity and polar interac-
tions, as well as decreasing logP.
We are currently developing
functional assays with these
compounds which will be re-
ported at a later date.
Figure 2. A) Structure of compound 5c, and binding directly to HumRADA2 (left, KD =35 mm) and showing no
binding to the wild-type RadA (right). B) Competitive ITC experiments to HumRADA2 indicate the binding site of
5c. Top row: ITC experiments in the absence of 5c determined the KD values of Ac-FHTA-NH2, the RadA oligomeri-
sation peptide, and ATP to be 290, 7.0, and 2.5 mm, respectively. Bottom row: ITC of Ac-FHTA-NH2, RadA oligomeri-
sation peptide, and ATP in the presence of 200 mm 5c shows competition with the FxxA binding site, but not the
ATP binding site.
Author contributions
D.E.S. and A.G.C. designed re-
search, performed research, ana-
lysed data, and wrote the paper.
tophan series, the LLE generally drops as the compounds are
M.H. designed research, performed research, and analysed data.
C.A., T.L.B., and A.V. designed research.
progressed from fragment 1 (LLE=1.9), to 6a (LLE=0.5) for
example. The situation is a little better for the sulfonamide
series; the most potent compound 7m (KD =1.3 mm) has an
LLE of 1.5. Compound 7p, which has a similar KD value of Acknowledgements
1.6 mm, is less lipophilic than 7m and has a marginally im-
proved LLE of 1.8. Taken together, the metrics indicate that al-
though the LE and LLE fall for the tryptophan series, the LLE
can be maintained in the sulfonamide series whilst improving
We thank May Marsh (Department of Biochemistry, University of
Cambridge) and David Dias (Department of Chemistry, University
of Cambridge) for useful discussions. We thank the Wellcome
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Published online on && &&, 0000
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FxxA in the crosshairs: Herein we
report two lead series of small mole-
cules chemically elaborated from
indole-containing fragments that bind
to the surface of a humanised RAD51
orthologue. Our most potent com-
pounds possess single-digit micromolar
potency and target the FxxA binding
site of the protein–protein interaction
with BRCA2.
D. E. Scott, A. G. Coyne, A. Venkitaraman,
T. L. Blundell, C. Abell, M. Hyvçnen*
&& – &&
Small-Molecule Inhibitors That Target
Protein–Protein Interactions in the
RAD51 Family of Recombinases
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