ACS Medicinal Chemistry Letters
Featured Letter
Cancer Institute, Boston, Massachusetts 02215, United States;
Harvard Medical School, Boston, Massachusetts 02115, United
Authors
Donald D. Raymond − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Christa A. Blomquist − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Charisse Flerida Pasaje − Department of Biological
Engineering, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, United States
Patrick R. McCarren − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Jamie Moroco − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States; orcid.org/
Figure 6. P. falciparum strain NF54 growth-inhibition assay. (A)
NF54 parasites were incubated with 1a, 1b, SLFb, or rapamycin in a
dose-dependent manner. Proliferation was measured by luminescence
after a complete life-cycle and normalized to DMSO (100%) and
chloroquine (100 nM, 0%). (B) Structural differences between
synthetic ligands and compound IC50s (μM).
Henock B. Befekadu − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Foxy P. Robinson − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Debjani Pal − Broad Institute of MIT and Harvard, Cambridge,
Massachusetts 02142, United States
Lisl Y. Esherick − Department of Biological Engineering,
Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, United States
Alessandra Ianari − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States
Jacquin C. Niles − Department of Biological Engineering,
Massachusetts Institute of Technology, Cambridge,
concentration range indicating that the Plasmodium assay data
are not a result of general cytotoxicity (Figure S7).
While the in vitro data confirm these compounds covalently
modify C106 and the NanoBRET assay demonstrates direct
target engagement of FKBP35 in a cellular context, additional
genetic studies will be necessary to fully establish whether
FKBP35 inhibition is responsible for their antiplasmodium
activity. Despite this limitation, our results suggest that
covalent inhibition of FKBP35 could be an avenue for the
development of antimalarial therapeutics. The presence of
C106 provides a functional handle for high-throughput
screening of covalent fragment libraries for the development
of novel chemical matter that could provide improved
selectivity between FKBP35 and the human FKBPs. While
we predominantly examined simple acrylamides, the slow rate
of covalent-bond formation indicates more electrophilic
warheads might prove beneficial for additional selectivity and
potency. In fact, switching to a more reactive acrylate
drastically accelerated the rate of covalent bond formation
but at the cost of plasma stability. Additional experiments
examining warhead reactivity should be a fruitful endeavor.
Alternatively, it is possible that the pipecolic acid core does not
provide an optimal foundation upon which to covalently attack
C106. Thus, screening efforts to uncover new high affinity
FKBP-binding chemotypes optimized for the covalent
modification strategy presented here have the potential to
create more potent and selective compounds for this highly
conserved enzyme. Such compounds will be of significant
interest for testing against liver-stage disease.
Complete contact information is available at:
Author Contributions
T.C.A., A.I., and W.R.S. generated the concept and experi-
ments. T.C.A. designed and synthesized all compounds,
analyzed data, and prepared the manuscript. D.D.R. prepared
and isolated recombinant proteins and ran DSC experiments
and mass spec assays. C.A.B. performed cell culture and
nanoBRET experiments. C.F.P. and L.Y.E. performed and
analyzed parasite viability experiments. P.R.M. performed
computational modeling. T.C.A. and H.B.B. performed FP
experiments. J.M. ran and with T.C.A. analyzed mass spec
experiments. F.P.R. and D.P. performed cell viability experi-
ments. A.I. and J.C.N. facilitated the collaboration. All authors
discussed the results and commented on the manuscript. All
authors have given approval to the final version of the
manuscript.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
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Funding
Synthesis and characterization of all compounds and
biophysical, biochemical, and biological assay proce-
This research was supported by The Broad Institute (SPARC
grant (T.C.A., D.D.R., W.R.S.) and Broad Next10 (J.C.N))
and The Bill and Melinda Gates Foundation (OPP1158199
and OPP1162467 (C.F.A.P., L.Y.E., J.C.N.)).
AUTHOR INFORMATION
Corresponding Authors
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Notes
The authors declare the following competing financial
interest(s): W.R.S. is a Board or SAB member and holds
equity in Peloton Therapeutics, Ideaya Biosciences, Civetta
Therapeutics, and Bluebird and has consulted for Array, Astex,
Dynamo Therapeutics, Ipsen, PearlRiver Therapeutics, Sanofi,
Thomas C. Atack − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States; orcid.org/
William R. Sellers − Broad Institute of MIT and Harvard,
Cambridge, Massachusetts 02142, United States; Dana-Farber
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ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX