Targeting glycolysis: A fragment based approach towards bifunctional inhibitors of hLDH-5

Article abstract of DOI:10.1039/c0cc01166e

hLDH-5 has emerged as a promising target for anti-glycolytic cancer chemotherapy. Here we report a first generation of bifunctional inhibitors, which show promising activity against hLDH-5.

Full text of DOI:10.1039/c0cc01166e

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Targeting glycolysis: a fragment based approach towards bifunctional
inhibitors of hLDH-5wz
Adam D. Moorhouse,a Christian Spiteri,a Pallavi Sharma,a Mire Zlohb and John E. Moses*a
Received 28th April 2010, Accepted 7th July 2010
DOI: 10.1039/c0cc01166e
hLDH-5 has emerged as a promising target for anti-glycolytic
cancer chemotherapy. Here we report a first generation of
bifunctional inhibitors, which show promising activity against
hLDH-5.
pathways, involved in the Warburg effect are still being
uncovered,6 hLDH-A and indeed the M4 isozyme (hLDH-5)
are primary targets for anti-glycolytic based therapeutic
intervention.7
We report here a new class of bifunctional ligands which
show selectivity towards hLDH-5. These novel compounds
were prepared using a fragment based ‘click’ chemistry8
approach, taking advantage of the high fidelity of the CuAAC
fusion reaction.9
The lactate dehydrogenase (LDH) enzyme catalyses the inter-
conversion of pyruvate and lactate with concomitant inter-
conversion of NADH and NAD+. It converts pyruvate, the
final product of glycolysis, to lactate when oxygen is absent or
in short supply, and it performs the reverse reaction during the
Cori cycle in the liver.1 At high concentrations of lactate, the
enzyme exhibits feedback inhibition and the rate of conversion
of pyruvate to lactate is decreased.
The structure of hLDH-5 cocrystallised with the active site
inhibitor oxamate and co-factor NADH has been solved,2
enabling the rational design of molecular inhibitors. Due to
the close proximity between substrate and co-factor binding
sites, we envisaged a fragment based approach could be
used to unify, through a triazole linkage, the substrate and
co-factor mimetics (Fig. 1).
In humans, hLDH exists as five tetrameric isozymes, desig-
nated hLDH-1 through 5, each formed from a combination of
two protein subunits M and H. hLDH-5 (M4) predominates in
the liver and skeletal muscle while hLDH-1 (H4) prevails in the
cardiac muscle, where it catalyses the reverse reaction using
lactate as an aerobic source of energy.2
In the design of our inhibitors, we required synthetically
viable ‘NADH-like’ fragments, and were drawn towards
bis(indolyl)maleimides (BIM’s) as potential candidates. BIM’s
are known adenosine mimetics and have been shown to be
potent inhibitors of NAD+ dependent sirtuins.10 Considering
their ready synthetic availability,11,12 opportunities for diversifi-
cation and validated binding, BIM’s were an excellent starting
point for our ligand design. Oxamate on the other hand is
a known inhibitor of LDH enzymes.13 Since oxamic acid
derivatives have also proven to inhibit pfLDH,14 we hypo-
thesised that N-alkylation to allow tethering would not adversely
affect binding interactions. Furthermore, we envisaged replacing
the oxamic acid functionality with a simple carboxylic acid
In tumour cells, an over-expression of oncogenes including
Ras, Src, and HER-2/Neu through stabilisation of HIF-1a
protein leads to an up-regulation of most proteins/enzymes
involved in the glycolytic pathway, including hLDH-5 and
the glucose transporters GLUT-1 and GLUT-3.3 This up-
regulation is found in greater than 70% of human cancer
cases, suggesting that tumour cells must acquire selective
advantages by shifting from normal glucose metabolism
(OXPHOS) to lactic acid production, even under normoxic
conditions, the so called glycolytic switch4 or Warburg effect.5
Recent gene knock-out experiments demonstrated that reduc-
tion in the hLDH-5 activity of neu-initiated mammary tumor
cells resulted in stimulation of mitochondrial respiration and
a decrease of mitochondrial membrane potential.3b It also
compromised the ability of these tumour cells to proliferate
under hypoxia.
The diminished tumorigenicity of these hLDH-5 deficient
cells could be reversed by complementation with the human
ortholog hLDH-A (gene coding for the M unit) and hLDH-5
(enzyme), thus demonstrating that hLDH-5 plays a key role in
tumour maintenance.3b Although the hierarchy of the possible
genetic alterations, and/or adoption of optimum metabolic
a School of Chemistry, University of Nottingham, University Park,
Nottingham, NG7 2RD, UK. E-mail: john.moses@nottingham.ac.uk;
Fax: +44 (0)115 95 13564; Tel: +44 (0)115 951 3533
b The School of Pharmacy, University of London, London,
WC1N 1AX, UK
w Electronic supplementary information (ESI) available: Extended
experimental data, experimental procedures and 1H and 13C NMR
spectra for all new compounds. See DOI: 10.1039/c0cc01166e
z This article is part of the ‘Emerging Investigators’ themed issue for
ChemComm.
Fig.
1 (A) Structure of the NADH cofactor and substrate,
(B) designed bifunctional inhibitor using BIM as adenosine mimetic
and oxamate-substrate mimetic tethered through a triazole linker.
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Scheme 1 Syntheses of ‘NADH-like’ and ‘substrate-like’ azido and alkyne functionalised fragments.
group. The tether would comprise alkyl chains of varying
length, fused through a triazole linker. Modelling studies using
the known crystal structure of the human lactate dehydrogenase
ternary complex with NADH and oxamate were performed
with hypothetical ligand 1, which appeared to bind appro-
priately across both the substrate and co-factor binding site
(see ESIw, Fig. S1).
Encouraged by these preliminary modelling studies, we
began the syntheses of the ‘NADH-like’ and ‘substrate-like’
binding moieties, containing complementary azide and alkyne
groups ready to be tethered using the CuAAC.8 Readily
available azide functionalised fragments, aliphatic carboxylic
acids 2–4,15 the aromatic azido carboxylic acids derivatives
5–8, and aromatic azido compounds 9–11 as potential sub-
strate binding mimetics were prepared (Scheme 1).16
A selection of alkyne functionalised BIM’s 12–15 were made
by condensation of the corresponding indole-3-acetamides
and indole-3-glyoxylates17 in the presence of tBuOK,12 as were
the indoyl heteroaryl maleimide (H-BIM) substrates 16–17,
using a similar condensation approach (Scheme 1). Uniting the
corresponding azido-substrate and alkynyl-co-factor mimetics
using microwave enhanced CuAAC16b gave a 31-member
library of 1,4-triazole fused ligands (18–48, see ESIw, Table S1)
of varied linker length/structure, in good yields (33–83%).18
Both the target ligands and fragments were screened for
hLDH-5 activity at relatively high concentrations (50 and
100 mg mLꢁ1) using a high-throughput 96-well plate UV assay,
where decrease in the concentration of NADH was measured
at 340 nm.19 The rate curves (DA/Dt) at these concentrations
were compared with those of a blank, and against the curve of
Fig. 2 Lead hit hLDH-5 bifunctional ligands 24, 29 and 31.
significant activity, whereas the BIM fragments were insoluble
in the assay medium and could not be evaluated.
The activities against three enzymes were tested, human hLDH-5
(liver), rabbit rLDH-5 (muscle) and bovine bLDH-1 (heart). The
IC50’s of the three lead compounds 24, 29 and 31 were measured
using the four-parameter logistic equation (GraphPad Prism for
Windows V5.02). The IC50 of sodium oxamate was also recorded
for comparative purposes (ESIw, Table S2). Interestingly, and
unlike sodium oxamate, none of the bifunctional compounds
(24, 29 and 31) demonstrated activity against the isozyme
bLDH-1, which is comprised exclusively of the LDH-B gene
product, the H-domain (H4). This unprecedented observation
was very promising and a step closer towards achieving selective
inhibition of hLDH-5, the major LDH in carcinoma. Such precise
targeting of the LDH-A gene product will be essential for future
development as an anti-cancer treatment.
the known inhibitor sodium oxamate (at 50 and 100 mg mLꢁ1
)
respectively. Compounds demonstrating inhibition more than/
or similar to sodium oxamate were identified and their IC50
subsequently evaluated. Three compounds (B10%), 24, 29
and 31, demonstrated promising activity at the concentration
tested and were taken forward for further study (Fig. 2).
Interestingly, the oxamate-like fragments did not show any
The IC50 values revealed that compounds 24 and 29 were
particularly promising lead structures, demonstrating notable
selectivity for the hLDH-5 isozyme over other isozymes tested.
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Relatively low micromolar IC50’s were exhibited for hLDH-5
(14.8 mM and 35.9 mM, respectively) (ESIw, Table S2), whereas
neither compound displayed inhibition of the bLDH-1 even at
high concentrations. The activity of 24 against hLDH-5 was
9-fold better than the standard sodium oxamate, making this
candidate a promising target for further development. It is
noteworthy that none of the bifunctional ligands bearing an
N-methyl indole showed any kind of activity against LDH
(ESIw, Table S1, 36, 37, 38, 39, 42, 43, 44, 45, 46). This may
imply a possible H-bonding interaction of the indole group of 24,
29 and 31 in the enzyme active site. Unfortunately, bifunctional
ligands bearing either a thiophene group (BIM fragment) or a
non-carboxylic group (azido-fragment) were insoluble in the
buffer medium (ESIw, Table S1, 25–27, 34, 35, 40–42, 47
and 48), and no conclusions could be drawn from these entries.
To help us understand the nature of ligand–target interaction,
docking experiments on the lead compounds were performed.
Of the three compounds, ligand 24 was the best fit for the
protein binding site, and overlayed well with the cofactor from the
crystal structure. A favourable interaction between the protein
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)
with hydrogen bonds formed between the protein and carboxylic
and BIM moiety (ESIw, Fig. S2). In line with the observed
IC50’s, ligands 29 and 31 were found to interact less favourably
(cf. 29 = ꢁ8.4 kcal molꢁ1 and 31 = ꢁ7.5 kcal molꢁ1).
Using a fragment based CC approach we have synthesised a
small library of bifunctional compounds, specifically designed to
target hLDH-5. We have identified three promising lead struc-
tures with impressive selectivity and activity. These ligands
represent a new generation of bifunctional inhibitors specifically
designed to target the hLDH-5 isozyme with a means to inter-
fering with tumour metabolism. Molecular docking experiments
corroborated our initial hypothesis and revealed favourable
interactions with both the substrate and co-factor binding sites.
Detailed molecular modelling investigations, and further
biological testing with a panel of cancer cell-lines are currently
underway and will be reported in due course.
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We thank EPSRC and The University of Nottingham for
financial support. Thanks to Dr K. Barral and Sally Dempster
for fruitful discussions.
18 Note: the products of the 31-member library were all isolated,
purified and characterised. Under the reaction conditions, some of
the fragments decomposed or the products could not be isolated or
adequately purified. In these cases, the samples were not taken
forward for screening.
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232 | Chem. Commun., 2011, 47, 230–232