DOI: 10.1002/cmdc.201600248
Full Papers
Substrate Fragmentation for the Design of M. tuberculosis
CYP121 Inhibitors
Madeline E. Kavanagh,[a] Janine L. Gray,[a] Sophie H. Gilbert,[a] Anthony G. Coyne,[a]
Kirsty J. McLean,[b] Holly J. Davis,[a] Andrew W. Munro,[b] and Chris Abell*[a]
The cyclo-dipeptide substrates of the essential M. tuberculosis
(Mtb) enzyme CYP121 were deconstructed into their compo-
nent fragments and screened against the enzyme. A number
of hits were identified, one of which exhibited an unexpected
inhibitor-like binding mode. The inhibitory pharmacophore
was elucidated, and fragment binding affinity was rapidly im-
proved by synthetic elaboration guided by the structures of
CYP121 substrates. The resulting inhibitors have low micromo-
lar affinity, good predicted physicochemical properties and se-
lectivity for CYP121 over other Mtb P450s. Spectroscopic char-
acterisation of the inhibitors’ binding mode provides insight
into the effect of weak nitrogen-donor ligands on the P450
heme, an improved understanding of factors governing
CYP121–ligand recognition and speculation into the biological
role of the enzyme for Mtb.
Introduction
Enzymes have evolved to bind substrates specifically. However,
the binding energetics of enzyme–substrate interactions are
optimised for catalytic efficiency, and therefore implicitly, they
should not bind too tightly. Consequently, the contribution to
the free energy of binding of individual structural motifs will
be unevenly distributed over the enzyme–substrate complex
and the relative importance of these interactions are difficult
to assess when combined in a large molecule.[1,2] In this way,
large (>250 Da) substrates are analogous to large molecule
drug leads or hits that are identified from a high throughput
screen. These compounds typically bind in the low micromolar
range by making multiple suboptimal interactions, and as
such, may not represent the best starting points for elabora-
tion.[3–7]
binding of a large substrate or a hit from a high throughput
screen. Fragment-based methods are now firmly established as
valuable techniques for identifying ligand efficient small mole-
cules that can act as leads for drug development.[13,14] The ap-
plication of fragment screening as a tool to assess target
druggability, identify binding hotspots, and interrogate biologi-
cal systems is also being increasingly appreciated.[15–17] The util-
ity of fragments for these applications is a consequence of
their small size and inherent ability to probe macromolecular
surfaces more effectively than larger compounds.[4,6]
The question as to whether fragments of a larger ligand
maintain the same binding interactions when they are discon-
nected has been addressed in recent years.[8–12,18–20] The size
and structures of the disconnected fragments, the choice of
disconnection, properties of the target macromolecule, and re-
quirements for cofactors or binding cooperativity, are all fac-
tors that have contributed to the variation in the results ob-
served. What is consistently apparent though, is that fragments
derived from large ligands bind preferentially to energetic hot-
spots and, regardless whether this binding mode recapitulates
that of the larger ligand, characterisation of these interactions
provides valuable insight for subsequent ligand design.[21,22]
Furthermore, lead deconstruction or fragmentation has result-
ed in the identification of novel chemical scaffolds for use as
drug leads and previously unknown binding sites, as well as
contributing to understanding the mechanisms of biomolecu-
lar recognition or catalysis; outcomes which are ultimately, of
more value.[8,9]
The deconstruction of large molecules into their representa-
tive fragments can provide an assessment of the binding con-
tributions of individual structural motifs, identify the minimal
pharmacophore required for biological activity and provide in-
sight into the fundamental basis of enzyme–substrate recogni-
tion.[8–12] This principle can be applied to deconvoluting the
[a] M. E. Kavanagh, J. L. Gray, S. H. Gilbert, Dr. A. G. Coyne, H. J. Davis,
Prof. C. Abell
Department of Chemistry, The University of Cambridge,
Lensfield Road, Cambridge, CB2 1EW (UK)
[b] Dr. K. J. McLean, Prof. A. W. Munro
Centre for Synthetic Biology of Fine and Specialty Chemicals
(SYNBIOCHEM), Manchester Institute of Biotechnology, Faculty of Life-
Sciences, The University of Manchester, Manchester M1 7DN (UK)
Although the retrospective deconstruction of successful
drug candidates, lead compounds and enzyme substrates has
been detailed in the literature,[9–12,19,23,24] there are few exam-
ples where the fragments derived in this way have been pur-
sued for subsequent ligand development.[25] Here, the dipep-
tide substrates of the cytochrome P450 enzyme CYP121 from
Supporting information for this article can be found under http://
ꢀ 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
any medium, provided the original work is properly cited.
ChemMedChem 2016, 11, 1924 –1935
1924 ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim