Structure-Guided Optimization of Replication Protein A (RPA)-DNA Interaction Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.9b00440

Replication protein A (RPA) is the major human single stranded DNA (ssDNA)-binding protein, playing essential roles in DNA replication, repair, recombination, and DNA-damage response (DDR). Inhibition of RPA-DNA interactions represents a therapeutic strategy for cancer drug discovery and has great potential to provide single agent anticancer activity and to synergize with both common DNA damaging chemotherapeutics and newer targeted anticancer agents. In this letter, a new series of analogues based on our previously reported TDRL-551 (4) compound were designed to improve potency and physicochemical properties. Molecular docking studies guided molecular insights, and further SAR exploration led to the identification of a series of novel compounds with low micromolar RPA inhibitory activity, increased solubility, and excellent cellular up-take. Among a series of analogues, compounds 43, 44, 45, and 46 hold promise for further development of novel anticancer agents.

Full text of DOI:10.1021/acsmedchemlett.9b00440

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Letter
Structure-guided Optimization of Replication
Protein A (RPA)-DNA Interaction Inhibitors.
Navnath S. Gavande, Pamela S VanderVere-Carozza, Katherine
S Pawelczak, Tyler L Vernon, Matthew Jordan, and John J. Turchi
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00440 • Publication Date (Web): 02 Jan 2020
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Structure-guided Optimization of Replication Protein A (RPA)-DNA
Interaction Inhibitors.
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Navnath S. Gavande,*,†,‡ Pamela S. VanderVere-Carozza,† Katherine S. Pawelczak,§ Tyler L. Vernon,†
Matthew Jordan,† and John J. Turchi*,†,§,⊥
†Department of Medicine, Indiana University School of Medicine (IUSM), Indianapolis, Indiana 46202 USA
‡Department of Pharmaceutical Sciences, Wayne State University College of Pharmacy and Health Sciences, Detroit, Michi-
gan 48201, USA
§NERx Biosciences, 212 W 10th St. Suite A480, Indianapolis, Indiana 46202, USA
⊥
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202,
USA
KEYWORDS: Replication Protein A, DNA damage and repair, reversible inhibitors, SAR, medicinal chemistry
ABSTRACT: Replication protein A (RPA) is the major human single stranded DNA (ssDNA)-binding protein, playing essential
roles in DNA replication, repair, recombination and the DNA-damage response (DDR). Inhibition of RPA-DNA interactions repre-
sents a therapeutic strategy for cancer drug discovery and has great potential to provide single agent anticancer activity and to syner-
gize with both common DNA damaging chemotherapeutics
and newer targeted anticancer agents. In this letter, a new
series of analogues based on our previously reported TDRL-
551 (4) compound were designed to improve potency and
physicochemical properties. Molecular docking studies
guided molecular insights and further SAR exploration led
to the identification of a series of novel compounds with low
micromolar RPA inhibitory activity, increased solubility and
excellent cellular up-take. Among a series of analogues,
compound 43, 44, 45 and 46 hold promise for further development of novel anti-cancer agents.
The clinical efficacy of many DNA damaging cancer
chemotherapeutics involves inducing DNA damage to push
cancer cells into apoptosis.1-2 DNA damage is repaired by in-
trinsic repair pathways and is coordinated by the DNA damage
response (DDR) pathway. One mechanism to enhance the ther-
apeutic window associated with these therapies is to direct treat-
ment to those cancers that harbor intrinsic DNA repair deficien-
cies.3-5 This approach has been effectively exploited in synthetic
lethal strategies to develop safe and effective treatments.1 With
an appropriately selected drug target, there is the potential to
enhance both single agent activity and synergistic anti-cancer
effect of the same chemical entity. Therefore, targeting DNA
repair and the DDR deficiencies has the potential for more
selective, better tolerated therapies to improve cancer pa-
tient survival.6-7
RPA-DNA interaction a promising target to develop novel anti-
cancer therapeutics. RPA is also overexpressed in a number of
cancers including lung, ovarian, breast, colon and esophageal11-
12 One rationale for targeting RPA is based on RPA exhaustion
which can lead to replication catastrophe. By extension, one
can envision that cancer cells exhibiting replication stress re-
quire more RPA for survival compared to non-cancer cells to
provide a therapeutic treatment window.
Over the past few years, several RPA inhibitors have been
identified and have either focusing on blocking the protein-pro-
tein13,15-22 or the protein-DNA interaction14,23-26 (Figure 1A).
Targeting the F-domain has proven effective for disrupting pro-
tein-protein interactions and the A and B domains of RPA70 for
protein-DNA inhibitors.27-29 Previously, we have successfully
discovered and developed compounds 1-4 which block the
RPA-DNA interaction.23-26 Particularly, compound 3 (TDRL-
505) and 4 (TDRL-551) display single agent activity in lung and
ovarian cancer cell lines and also synergize with cisplatin and
Replication protein A (RPA) is the major human ssDNA
binding protein and plays an integral role in both nucleotide ex-
cision repair (NER) and homologous recombination (HR) DNA
repair pathways in addition to its essential role in DNA replica-
tion and DDR.8-10 Each of these critical roles of RPA make the
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Figure 1. A) Structure of previously reported RPA inhibitors targeting RPA-DNA interaction.23-26 B) Schematic representation of SAR
design rationale: a large space-filling pocket surrounding Ring A of compound 4 and amino acid residues surrounding alkyl carboxylic acid
side chain which can be extended towards the solvent-exposed region for further structural optimization. [Compound 4 hydrogen bond
interaction with Ser392 and Arg382 is indicated with the dashed magenta line, π-π stacking interactions with Trp361 and Phe386 are shown
in solid magenta dumbbell, and salt-bridge interactions with Lys313 are shown in dashed two-sided magenta arrow.]
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etoposide.25-26 The mechanism of action of both compounds is
via a reversible interaction with the central OB-folds in DNA
binding domains A and B of RPA70. In vivo analysis of com-
pound 4 (TDRL-551) revealed minimal cytotoxicity alone and
in combination with platinum was able to significantly delay
tumor growth in a NSCLC xenograft model.26 Both compounds
3 and 4, however, had limited solubility and cell permeability.
In this Letter, we further expanded our structure-guided drug
design efforts by exploiting compound 4 (TDRL-551) scaffold
with the aim to improve RPA inhibitory potency, solubility and
cellular uptake.
18 were obtained in good yields by acylation at N1 of the pyra-
zoline ring of compounds 15a-d with glutaric anhydride in
chloroform. Upon reduction of nitro group of 18 by stannous
chloride, the desired amine product 19 was obtained in an ac-
ceptable yield. The synthesis of compound 20 and 21 was ac-
complished by acylation of hydroxyl group of compound 17 us-
ing acryloyl chloride and morpholinecarbonyl chloride, respec-
tively, in basic condition. To further extend the substitution on
para-position at ring A, we have synthesized target compounds
22-29 as depicted in Scheme 1 and supplemental Scheme S1.
Compound 30 was prepared from an above synthesized precur-
sor 15a by N1-acylation of the pyrazoline ring using ethyl glu-
taryl chloride in basic condition. Intermediates 31a-d were pre-
pared by optimizing Pd-catalyzed Suzuki coupling of com-
pound 30 with corresponding boronic acids/esters using non-
aqueous CsF in dimethoxyethane protocol. Final target com-
pounds 22-25 were obtained from compounds 31a-d by hydrol-
ysis of corresponding alkyl esters using 10N NaOH at room
temperature. Our initial attempt utilizing Scheme 1 to synthe-
size halopyridine containing compounds 26-28 had limited ef-
ficiency as a function of the late stage Suzuki coupling reaction.
Therefore, we have achieved efficient synthesis of target com-
pounds 26-29 as outlined in Supplemental Scheme S1. Early
stage Suzuki coupling of 4′-iodoacetophenone 7 with commer-
cially available boronic acids/esters 32a-d catalyzed by
tetrakis(triphenylphosphine)palladium(0) in the presence of
aqueous potassium carbonate base provided precursors 33a-d.
1,3-diarypropenones 34a-d were synthesized using an above
described Claisen-Schmidt condensation of corresponding sub-
stituted methyl ketones 33a-d with quinoline carbaldehyde 6
then 1,3-diarypropenones 34a-d refluxed with hydrazine hy-
drate in ethanol to afford corresponding 2-pyrazolines 35a-d in
good yields. Finally, acylation at N1 of the pyrazoline ring of
compounds 35a-d with glutaric anhydride in chloroform under
reflux provided target compounds 26-29 in moderate to good
yields after recrystallization in ethanol.
Initial molecular docking studies with compounds 3 and 4
revealed that both compounds have a high predicted affinity for
DNA binding domain B. We performed molecular docking
studies mainly focusing on the central DNA binding domains A
and B of RPA70 by using RPA70181-422 X-ray crystal structure
(PDB code: 1FGU).29 The interaction presented in the Figure
1B reveals the stability is driven via hydrophobic and π-π inter-
actions, and that the conserved alkyl carboxylic acid side chain
is stabilized via interactions with basic amino acid residues
which are critical for inhibitory activity. In addition, docking
studies revealed the extended terminal carboxylic acid side
chain appeared to orient towards the solvent-exposed region of
the protein and provides a potential site for modulating the
physicochemical properties of these compounds. We have ex-
ploited compound 4’s interaction with domain B to outline a
SAR design for further structural optimization (Figure 1B). We
first pursued optimizing aromatic Ring A exploiting the large
pocket around it and also simultaneously optimizing alkyl car-
boxylic acid side chain to improve the drug-like properties.
The synthesis of target compound 4 and its analogs 16-25 is
depicted in Scheme 1. We have obtained compound 4 and 16-
25 from starting material 3-ethoxyaniline by slightly modifying
our previous synthetic protocol.26 The requisite quinoline
carbaldehyde 6 was prepared by acylation of 3-ethoxyaniline
with acetic anhydride, followed by multicomponent reaction
that involves process of Vilsmeier-Haack chlorination,
formylation and cyclization of acetanilide using DMF and
POCl3. Claisen-Schmidt condensation of corresponding substi-
tuted acetophenones 7-10 with quinoline carbaldehyde 6 in the
presence of aqueous NaOH in ethanol yielded corresponding
1,3-diarypropenones 11-14 which on further treatment with hy-
drazine hydrate in ethanol under reflux afforded the correspond-
ing 2-pyrazolines 15a-d in good yields. Compound 4 and 16-
To optimize the alkyl carboxylic acid side chain of compound
4, we have synthesized compounds 37-47 as depicted in Sup-
plemental Schemes S2 and S3. The N1-acylation of the pyra-
zoline ring of precursor 15a using alkyl acyl chloride in basic
conditions afforded ester derivatives 36a-b and subsequent es-
ter hydrolysis using NaOH, provided analogs 37-38 in excellent
yields (Supplemental Scheme S2). To introduce rigidity in the
RPA binding pocket, we have synthesized compounds 39-40
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Scheme 1. Synthesis of Compound 4 and Analogs 16-25a
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aReagents and conditions: (a) acetic anhydride, DIPEA, DMAP, DCM, rt for 2 h, 88%; (b) (i) DMF, POCl3, 0°C for 25 min, (ii) acetanilide
5, 110°C for 3 h, 74%; (c) 2.5M NaOH, EtOH, 45°C for 45 min to 1 hr, 53-74%; (d) hydrazine hydrate, EtOH, reflux for 2-3 h, 70-83%; (e)
glutaric anhydride, CHCl3, reflux for 2 h, 58-71% (after recrystallization); (f) SnCl2, EtOH:THF (1:1), reflux for 2 h, 48% (after recrystalli-
zation); (g) acryloyl chloride, 2N NaOH, THF, 0°C to rt for 2 h, 34%; (h) 4-morpholinecarbonyl chloride, TEA, DMAP, THF, 0°C to rt for
12 h, 63%. (i) ethyl glutaryl chloride, DIPEA, DCM, rt for 12 h, 78%; (j) boronic acid/ester, Pd(PPh3)4, CsF, DME, 90°C for 15-18 h, 63-
69%; (k) 10N NaOH, THF:MeOH (1:2), rt for 6-8 h, 75-87%.
from precursor 11 and using corresponding substituted hydra-
zinobenzoic acid under reflux condition in AcOH and n-buta-
nol. Furthermore, the morpholine/morpholinoalkylamine
group was attached to the oxopentanoic acid side chain of cor-
responding compound 4 or 16 or 26 or 28 or 29 by utilizing
EDCI/HOBt amide synthetic protocol to achieve final com-
pounds 41-47 in good yields (Supplemental Scheme S3).
(IC50 = 6.7 ± 0.64 μM). Insertion of another heteroatom such
as nitrogen into the furan ring in the form of an isoxazole dis-
played even more potent RPA inhibitory activity (compound
24, IC50 = 1.7 ± 0.28 μM). The difference in activity with the
small changes of heteroatom within 5-membered ring may be
due to the orientation of the ring or ability of the heteroatom
to interact with RPA backbone binding pocket amino acid res-
idues. Further extension of compound 4 Ring A in the space-
filling binding cleft with a substituted bulky biphenyl het-
eroaromatic ring such as halogenated pyridinyl compounds
(26-29) exhibited even more potent RPA inhibitory activities
than parent compound with an increased potency between 4-
and 15-fold. Among all the Ring A modifications, chloro-pyr-
idinyl compound 26 and bromo-pyridinyl compound 27
showed the most potent RPA inhibitory activity in the EMSA
assay (26, 1.6 ± 0.04 μM and 27, IC50 = 1.0 ± 0.60 μM). Fur-
ther, we have included fluorine and trifluoromethyl substitu-
The assessment of RPA inhibitory activity was performed
using a highly sensitive and quantitative electrophoretic mo-
bility shift assay (EMSA). The assay employs purified het-
erotrimeric human RPA devoid of any tags or modifications
and measures the impact of the compounds on direct RPA
binding to a ss-DNA substrate.23,26,30 We also have confirmed
that the compounds specifically target OB-folds A and B us-
ing an RPA construct consisting of this minimal DNA binding
domain (See Supplemental Table S2), as we have previously
described.25 RPA can also interact with damage duplex DNA
via binding to the undamaged ssDNA regions opposite the
damage.30 The RPA inhibitory compounds also, as expected,
block this interaction. For the purpose of quantifying RPA
inhibitory activity we used gold-standard, full length RPA
binding to single stranded DNA to calculated IC50 values of
newly synthesized analogs and provide insight into the struc-
ture-activity relationships (Table 1 and 2). The replacement
of the iodo group of compound 4 (IC50 = 15.3 ± 1.42 μM) with
hydroxyl or nitro groups resulted in a near complete loss of
RPA inhibitory activity while changing to an amine resulted
in a 7-fold increase in potency (IC50 = 2.3 ± 0.22 μM). Acry-
late containing compound 20 exhibited 2-fold increase in ac-
tivity compared to 4, surprisingly, the inclusion of mor-
pholinecarbonyl group in compound 21 resulted in poor RPA
inhibition. Introduction of a heteroaromatic on Ring A such as
3’-furan, 2’-furan and 4’-isoxazole boosted potency drasti-
cally as 2’-substituted furan at Ring A (compound 23, IC50
= 2.1 ± 0.48 μM) exhibited almost 3.2-fold increase in activity
compared to 3’-substituted furan containing compound 22
ents in pyridinyl compounds 28-29. However, both com-
pounds displayed slightly weaker activity compared to chloro-
and bromo-pyridinyl containing compounds 26-27. These
data revealed the importance of bulky hydrophobic substitu-
ents on Ring A of compound 4 to occupy space-filling binding
pocket of RPA and are consistent with our postulated molec-
ular docking binding pockets outlined in an above SAR design
section.
The next aim was to explore SAR on the alkyl carboxylic acid
side chain of compound 4, with a focus on enhancing physi-
cochemical properties while retaining or improving potency.
Modification of alkyl side chain of compound 4 revealed a
series of interesting activity against RPA (Supplemental Ta-
ble S1). The conversion to an oxopentanoic ethyl ester (com-
pound 30) completely abrogated RPA inhibitory activity. A
similar trend was observed with compounds 39-40 when we
replaced oxopentanoic acids with 2’ or 3’-subsitituted ben-
zoylic carboxylic acids. The intramolecular distances between
the benzoylic carboxylic acids and the core dihydropyrazole
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As discussed earlier, initial docking study positioned the terminal
Table 1. SAR of Ring A with RPA IC50 Values of Analogs
16-29a
carboxylic group towards the solvent-exposed region of the pro-
tein and could provide a good potential for modulating the phys-
icochemical properties of our compounds. In addition, carboxylic
acid functional group often have limited utility in drug discovery
due to limited passive diffusion across biological membranes
(cellular permeability) and metabolic instability.31 Therefore, we
articulated that RPA inhibitors could be improved by introducing
an additional solubilizing group into the terminal carboxylic acid
group without significantly influencing the RPA inhibitory activ-
ities. To this end, the water-soluble morpholine moiety was em-
ployed to enhance the physicochemical properties. Consistent
with our docking analysis, replacement of the terminal carboxylic
group with morpholinoethane (compound 42) and morpho-
linopropane (compound 43), retained the inhibitory activity while
compound 43 showed an almost 3-fold increase in potency (IC50
= 4.9 ± 0.28 μM) compared to our parent compound 4. Encour-
aged by these results, a series of new analogs (Table 2, com-
pounds 44-47) were further designed and synthesized by incor-
porating morpholinopropane at the terminal carboxylic group of
compound 16, 26 and 28-29. All these compounds exhibited
modest to potent RPA inhibitory activity compared to the parent
compound 4 and anticipated to improve physiochemical proper-
ties for better cellular and in vivo activities.
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Cpd
4
R1
RPA IC50 (μM)b,c
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I
15.3 ± 1.42
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Br
19.2 ± 4.17
>25
OH
NO2
NH2
>25
2.3 ± 0.22
8.9 ± 0.98
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>25
Table 2. SAR of Side Chain Modifications with RPA IC50
Values of Analogs 41-47a
6.7 ± 0.64
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1.6 ± 0.04
1.0 ± 0.60
Cpd R1
R2
RPA IC50 (μM)b,c
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I
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10.5 ± 0.68
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2.6 ± 0.60
4.3 ± 0.42
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I
4.9 ± 0.28
10.0 ± 1.46
4.0 ± 0.56
2.3 ± 0.31
5.8 ± 0.39
Br
aDetermined using EMSA, binding of full length human RPA
to DNA was assessed. Compounds that displayed greater than
80% inhibition at 25 μM were analyzed in titration experiments.
cIC50 values are a mean of minimum of triplicate independent ex-
periments and data are presented as the mean ± SD.
b
were very similar comparing to the oxopentanoic acids. The
dramatic reduction in activity of the benzoylic carboxylic acid
derivatives is likely function on the rigidity induced restrict-
ing motion of the molecule within the RPA-DNA binding
pocket independent of position of the carboxylic acid. Increas-
ing the length of the aliphatic side chain by 1 and 2 carbon, in
compounds 37 and 38 respectively, had only modest effects
on potency and similar inhibition was observed by direct ad-
dition of a N-morpholino group on alkyl carboxylic acid side
chain depicted in compound 41
aDetermined using EMSA, binding of full length human RPA
b
to DNA was assessed. Compounds that displayed greater than
80% inhibition at 25 μM were analyzed in titration experiments.
cIC50 values are a mean of minimum of triplicate independent ex-
periments and data are presented as the mean ± SD.
To confirm the mechanism of inhibitions via compound binding
to the target protein and not via binding to the DNA substrates,
we conducted a fluorescent intercalator displacement (FID) assay
as described previously.32 The displacement of a DNA binding
dye and decrease in fluorescence is indicative of the compound’s
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biochemical in vitro IC50.25-26 This suggested cellular uptake
could be limiting cellular activity. The charge on the carbox-
ylic acid, while potentially increasing aqueous solubility, was
also anticipated to negatively impact cellular uptake. We
therefore assessed aqueous solubility of a series of the most
biochemically active compound 4 derivatives. Figure 4A
shows data obtained with assessing aqueous solubility in un-
buffered H2O at pH 7. The data reveal a general trend of the
morpholino modified compounds displaying increased solu-
bility compared to their carboxylic acid counterparts (com-
pounds 4, 16, 26, 28, 29 Vs 43, 44, 45, 46, 47, respectively).
ability to bind DNA. In order to probe the potential role of DNA
intercalation as a mechanism for RPA inhibition, most potent
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compounds 19, 23, 26, 27, 43 and 45 were analyzed using FID
assay along with doxorubicin (Dox) as a positive control. The re-
sults presented in Figure 2 demonstrate as expected that positive
control, doxorubicin a known non-covalent DNA binding chemo-
therapeutic, resulted in a concentration dependent reduction in
fluorescence. As expected, minimal DNA binding activity was
observed for any of the novel RPA inhibitors (19, 23, 26, 27, 43
and 45). The slight reduction in signal cannot account for the
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Figure 2. Analysis of compound interactions with DNA using
Fluorescent intercalator displacement (FID) assay. The indicated
concentrations of doxorubicin (Dox), compounds 19, 23, 26, 27,
43 and 45 were analyzed for the ability to displace a fluorescent
Sybr-green DNA intercalator as a measure of compound DNA
interactions. The data represent the average and SD of three in-
dependent experimental determinations performed in duplicates.
Figure 3. Molecular docking studies (PDB code: 1FGU)29: A-C)
Molecular interactions of compound 26 (A), 42 (B) and 45 (C)
(all in yellow carbon) with hRPA (key amino acids are shown in
dark cyan carbon and cartoon is shown in pale cyan color).
Interaction with amino acid side chains is indicated with the
dashed magenta lines, π – π stacking interactions are shown in
solid magenta dumb ell and salt-bridge interactions are shown in
dashed two-sided magenta arrow. Interaction distances indicated
in Å. D) Molecular overlay (superimposition) of compound 26,
42 and 45 (all in yellow carbon) in the RPA binding site.
high potency of the compounds. These data suggest that the
mechanism of action involves targeting the protein- DNA
interaction by directly binding to the RPA protein. Consistent
with this conclusion is the lack of inhibitory agains other OB-fold
containing proteins
The cellular uptake or intracellular metabolism of the
compounds plays an important role in cellular efficacy and it
allows to properly rationalize structure-activity relationships
to enhance the sustained therapeutic effect. Therefore, we
evaluated cellular uptake of our lead compounds in H460
NSCLC cells (Figure 4B). The data demonstrate that as ex-
pected that compound 4 has relatively poor uptake while com-
pound 26 showed increased uptake even with the carboxylic
acid moiety. The morpholino derivative of these both com-
pounds demonstrated considerably superior uptake observed
with the compounds 43 and 45. We expect the increased up-
take will increase cellular RPA inhibition and anti-cancer ac-
tivity. A similar trend of increased cellular uptake was also
observed in morpholino modified compound 46 and 47 com-
pared to their carboxylic acid counterparts (Data not shown).
To delineate the key interactions and to understand the SAR
of newly synthesized compounds, these compounds were flexibly
docked mainly focusing on the central DNA binding domains A
and B of RPA70 as discussed above.29 All newly developed
inhibitors docked well at this site. Figure 3 shows the binding
orientation and molecular interactions of representative
compound 26, 42 and 45 within the RPA domain B region. The
molecular interaction of 26, 42 and 45 (Figures 3A-C,
respectively) is largely ascribed to various electrostatic
interactions, including, i) compound 42 and 45 amide carbonyl
make hydrogen bond contacts with the amine of Lys313, while
compound 26 terminal carboxylic acid makes salt-bridge
interactions with the Lys313; ii) amide carbonyl (attached to
pyrazole ring) of all three compounds make strong hydrogen
bond contacts with the hydroxyl group of Ser392 as well as with
the amine group of Arg382; iii) the π – π stacking interactions
between the phenyl moiety (Ring A) and the aromatic ring of
Trp361 in all three compounds. In addition, all three compounds
quinoline moiety is also well positioned to make π – π stacking
interactions with Phe386; iv) Terminal alkyl morpholino side
chain is well fitted and located as its extending out of the RPA
binding region into a solvent exposed region. Docking studies
predicted a stronger affinity of the compound 26 than other series
of compounds including compound 42 and 45, in fact compound
26 also showed potent RPA inhibition than other series of
compounds in in-vitro EMSA assay (Table 1).
Figure 4. Solubility analysis and cellular uptake of RPA inhibi-
tors. A) Aqueous solubility and B) Cellular uptake of representa-
tive compounds were determined as described in the “Supporting
Our previous data demonstrated that compound 3 and 4
displayed a significantly higher cellular IC50 compared to the
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Information”. Individual data points are plotted and bars repre-
sent the mean and SD of three independent experimental deter-
minations.
DDR, DNA Damage Response; NER, Nucleotide Excision
Repair; RPA, Replication Protein A; DBD, DNA Binding Do-
main; HOBt, Hydroxybenzotriazole; EDCI, 1-Ethyl-3-(3-(di-
methylamino)propyl)-carbodiimide; DIPEA, N,N-Diisopro-
pylethylamine; DMAP, 4-Dimethylaminopyridine; DCM, Di-
chloromethane; DMF, N,N-Dimethylformamide; THF, Tetra-
hydrofuran; TEA, Triethylamine; DME, Dimethoxyethane;
EMSA, Electrophoretic Mobility Shift Assay; SAR, Structure
Activity Relationship; FID, Fluorescent Intercalator Displace-
ment; Dox, Doxorubicin
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In conclusion, we have extended SAR study of our previ-
ously reported RPA inhibitor by utilizing a structure-based
drug design strategy. We have identified a series of novel
chemical inhibitors that interact directly with RPA to block its
interaction with single-stranded DNA and most importantly
these inhibitors do not bind directly with DNA. The system-
atic SAR exploration had revealed that a heteroaromatic ring
or reasonably a larger lipophilic biphenyl ring at the Ring A
and an extension of the terminal carboxylic acid side chain are
well tolerated. Particularly, the introduction of morpholino
group at the terminal alkyl carboxylic acid side chain resulted
in the enhanced potency, solubility and cellular up-take. Com-
pound 43, 45 and 46 represent excellent lead compounds with
drug-like properties suitable for future cellular and in vivo
studies. These efforts will be addressed in a subsequent man-
uscript.
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ASSOCIATED CONTENT
Supporting Information
Supplementary Figures S1, synthetic Schemes S1-S3, Table S1,
and Table S2, synthetic experimental procedures along with char-
acterization data, biological and physiochemical experimental
procedures, copies of 1H NMR spectra of final compounds, mo-
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The Supporting Information is available free of charge on the
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Conflict of Interest Disclosure
J Turchi is a co-founder of NERx Biosciences and receives re-
search funding from NERx Biosciences.
AUTHOR INFORMATION
Corresponding Authors
*John J. Turchi: E-mail Address: jturchi@iu.edu (JJT). Tel.:
+1-(317)-278-1996; fax, +1-(317)-274-0396.
*Navnath S. Gavande: E-mail Address: ngavande@wayne.edu
(NSG). Tel.: +1-(313)-577-1523; fax, +1-(313)-577-2033.
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ORCID
Navnath S. Gavande: 0000-0002-2413-0235
John J. Turchi: 0000-0001-5375-2992
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Author Contributions
The manuscript was written by N.S.G. and J.J.T. with editing
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given approval to the final version of the manuscript.
Funding Sources
This work is supported by NIH grants R01-CA180710 and R41-
CA162648 and the Tom and Julie Wood Family Foundation.
ACKNOWLEDGMENT
We thank Dr. Lifan Zeng and Erica Woodall for technical assis-
tance with HPLC and mass spectrometry (LCMS and HRMS).
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