10 research outputs found
Strategy to Target the Substrate Binding site of SET Domain Protein Methyltransferases
Protein
methyltransferases (PMTs) are a novel gene family of therapeutic
relevance involved in chromatin-mediated signaling and other biological
mechanisms. Most PMTs are organized around the structurally conserved
SET domain that catalyzes the methylation of a substrate lysine. A
few potent chemical inhibitors compete with the protein substrate,
and all are anchored in the channel recruiting the methyl-accepting
lysine. We propose a novel strategy to design focused chemical libraries
targeting the substrate binding site, where a limited number of warheads
each occupying the lysine-channel of multiple enzymes would be decorated
by different substituents. A variety of sequence and structure-based
approaches used to analyze the diversity of the lysine channel of
SET domain PMTs support the relevance of this strategy. We show that
chemical fragments derived from published inhibitors are valid warheads
that can be used in the design of novel focused libraries targeting
other PMTs
Evidence That Compound I Is the Active Species in Both the Hydroxylase and Lyase Steps by Which P450scc Converts Cholesterol to Pregnenolone: EPR/ENDOR/Cryoreduction/Annealing Studies
Cytochrome
P450scc (CYP 11A1) catalyzes the conversion of cholesterol
(Ch) to pregnenolone, the precursor to steroid hormones. This process
proceeds via three sequential monooxygenation reactions: two hydroxylations
of Ch first form 22(<i>R</i>)-hydroxycholesterol (HC) and
then 20α,22(<i>R</i>)-dihydroxycholesterol (DHC);
a lyase reaction then cleaves the C20–C22 bond to form pregnenolone.
Recent cryoreduction/annealing studies
that employed electron paramagnetic resonance (EPR)/electron nuclear
double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) <i>J. Am. Chem. Soc. 134</i>, 17149] showed that compound I (Cpd
I) is the active intermediate in the first step, hydroxylation of
Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize
the intermediates in the second and third steps of the enzymatic process,
as conducted by 77 K radiolytic one-electron cryoreduction and subsequent
annealing of the ternary oxy-cytochrome P450scc complexes with HC
and DHC. This procedure is validated by showing that the cryoreduced
ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically
competent and during annealing generate DHC and pregnenolone, respectively.
Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K
produces the superoxo-ferrous P450scc intermediate along with a minor
fraction of ferric hydroperoxo intermediates. The superoxo-ferrous
intermediate converts into a ferric-hydroperoxo species after annealing
at 145 K. During subsequent annealing at 170–180
K, the ferric-hydroperoxo intermediate converts to the
primary product complex with the large solvent kinetic isotope effect
that indicates Cpd I is being formed, and <sup>1</sup>H ENDOR measurements
of the primary product formed in D<sub>2</sub>O demonstrate that Cpd
I is the active species. They show that the primary product contains
Fe(III) coordinated to the 20-O<sup>1</sup>H of DHC with the <sup>1</sup>H derived from substrate, the signature of the Cpd I reaction.
Hydroperoxo ferric intermediates are the primary species formed during
cryoreduction of the oxy-P450scc-DHC
ternary complex, and they decay at 185 K with a strong solvent kinetic
isotope effect to form low-spin ferric
P450scc. Together, these observations indicated that Cpd I also is
the active intermediate in the C20,22 lyase final step. In combination
with our previous results, this study thus indicates that Cpd I is
the active species in each of the three sequential monooxygenation
reactions by which P450scc catalytically converts Ch to pregnenolone
Discovery of a Potent and Selective Coactivator Associated Arginine Methyltransferase 1 (CARM1) Inhibitor by Virtual Screening
Protein
arginine methyltransferases (PRMTs) represent an emerging
target class in oncology and other disease areas. So far, the most
successful strategy to identify PRMT inhibitors has been to screen
large to medium-size chemical libraries. Attempts to develop PRMT
inhibitors using receptor-based computational methods have met limited
success. Here, using virtual screening approaches, we identify 11
CARM1 (PRMT4) inhibitors with ligand efficiencies ranging from 0.28
to 0.84. CARM1 selective hits were further validated by orthogonal
methods. Two structure-based rounds of optimization produced <b>27</b> (SGC2085), a CARM1 inhibitor with an IC<sub>50</sub> of
50 nM and more than hundred-fold selectivity over other PRMTs. These
results indicate that virtual screening strategies can be successfully
applied to Rossmann-fold protein methyltransferases
Exploiting an Allosteric Binding Site of PRMT3 Yields Potent and Selective Inhibitors
Protein arginine
methyltransferases (PRMTs) play an important role
in diverse biological processes. Among the nine known human PRMTs,
PRMT3 has been implicated in ribosomal biosynthesis via asymmetric
dimethylation of the 40S ribosomal protein S2 and in cancer via interaction
with the DAL-1 tumor suppressor protein. However, few selective inhibitors
of PRMTs have been discovered. We recently disclosed the first selective
PRMT3 inhibitor, which occupies a novel allosteric binding site and
is noncompetitive with both the peptide substrate and cofactor. Here
we report comprehensive structure–activity relationship studies
of this series, which resulted in the discovery of multiple PRMT3
inhibitors with submicromolar potencies. An X-ray crystal structure
of compound <b>14u</b> in complex with PRMT3 confirmed that
this inhibitor occupied the same allosteric binding site as our initial
lead compound. These studies provide the first experimental evidence
that potent and selective inhibitors can be created by exploiting
the allosteric binding site of PRMT3
Discovery of a Potent, Selective, and Cell-Active Dual Inhibitor of Protein Arginine Methyltransferase 4 and Protein Arginine Methyltransferase 6
Well-characterized
selective inhibitors of protein arginine methyltransferases
(PRMTs) are invaluable chemical tools for testing biological and therapeutic
hypotheses. Based on <b>4</b>, a fragment-like inhibitor of
type I PRMTs, we conducted structure–activity relationship
(SAR) studies and explored three regions of this scaffold. The studies
led to the discovery of a potent, selective, and cell-active dual
inhibitor of PRMT4 and PRMT6, <b>17</b> (MS049). As compared
to <b>4</b>, <b>17</b> displayed much improved potency
for PRMT4 and PRMT6 in both biochemical and cellular assays. It was
selective for PRMT4 and PRMT6 over other PRMTs and a broad range of
other epigenetic modifiers and nonepigenetic targets. We also developed <b>46</b> (MS049N), which was inactive in biochemical and cellular
assays, as a negative control for chemical biology studies. Considering
possible overlapping substrate specificity of PRMTs, <b>17</b> and <b>46</b> are valuable chemical tools for dissecting specific
biological functions and dysregulation of PRMT4 and PRMT6 in health
and disease
Discovery of a Potent Class I Protein Arginine Methyltransferase Fragment Inhibitor
Protein
methyltransferases (PMTs) are a promising target class
in oncology and other disease areas. They are composed of SET domain
methyltransferases and structurally unrelated Rossman-fold enzymes
that include protein arginine methyltransferases (PRMTs). In the absence
of a well-defined medicinal chemistry tool-kit focused on PMTs, most
current inhibitors were identified by screening large and diverse
libraries of leadlike molecules. So far, no successful fragment-based
approach was reported against this target class. Here, by deconstructing
potent PRMT inhibitors, we find that chemical moieties occupying the
substrate arginine-binding site can act as efficient fragment inhibitors.
Screening a fragment library against PRMT6 produced numerous hits,
including a 300 nM inhibitor (ligand efficiency of 0.56) that decreased
global histone 3 arginine 2 methylation in cells, and can serve as
a warhead for the development of PRMT chemical probes
Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1)
WD
repeat-containing protein 5 (WDR5) is an important component
of the multiprotein complex essential for activating mixed-lineage
leukemia 1 (MLL1). Rearrangement of the MLL1 gene is associated with
onset and progression of acute myeloid and lymphoblastic leukemias,
and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics.
Our previous work showed that binding of small molecule ligands to
WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase
activity. Initial structure–activity relationship studies identified <i>N</i>-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides
as potent and selective antagonists of this protein–protein
interaction. Guided by crystal structure data and supported by in
silico library design, we optimized the scaffold by varying the C-1
benzamide and C-5 substituents. This allowed us to develop the first
highly potent (<i>K</i><sub>disp</sub> < 100 nM) small
molecule antagonists of the WDR5-MLL1 interaction and demonstrate
that <i>N</i>-(4-(4-methylpiperazin-1-yl)-3′-(morpholinomethyl)-[1,1′-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide <b>16d</b> (OICR-9429) is a potent and selective chemical probe suitable
to help dissect the biological role of WDR5
Structure-Based Optimization of a Small Molecule Antagonist of the Interaction Between WD Repeat-Containing Protein 5 (WDR5) and Mixed-Lineage Leukemia 1 (MLL1)
WD
repeat-containing protein 5 (WDR5) is an important component
of the multiprotein complex essential for activating mixed-lineage
leukemia 1 (MLL1). Rearrangement of the MLL1 gene is associated with
onset and progression of acute myeloid and lymphoblastic leukemias,
and targeting the WDR5-MLL1 interaction may result in new cancer therapeutics.
Our previous work showed that binding of small molecule ligands to
WDR5 can modulate its interaction with MLL1, suppressing MLL1 methyltransferase
activity. Initial structure–activity relationship studies identified <i>N</i>-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides
as potent and selective antagonists of this protein–protein
interaction. Guided by crystal structure data and supported by in
silico library design, we optimized the scaffold by varying the C-1
benzamide and C-5 substituents. This allowed us to develop the first
highly potent (<i>K</i><sub>disp</sub> < 100 nM) small
molecule antagonists of the WDR5-MLL1 interaction and demonstrate
that <i>N</i>-(4-(4-methylpiperazin-1-yl)-3′-(morpholinomethyl)-[1,1′-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide <b>16d</b> (OICR-9429) is a potent and selective chemical probe suitable
to help dissect the biological role of WDR5
Synthesis, Optimization, and Evaluation of Novel Small Molecules as Antagonists of WDR5‑MLL Interaction
The WD40-repeat protein WDR5 plays a critical role in
maintaining
the integrity of MLL complexes and fully activating their methyltransferase
function. MLL complexes, the trithorax-like family of SET1 methyltransferases,
catalyze trimethylation of lysine 4 on histone 3, and they have been
widely implicated in various cancers. Antagonism of WDR5 and MLL subunit
interaction by small molecules has recently been presented as a practical
way to inhibit activity of the MLL1 complex, and <i>N</i>-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides were
reported as potent and selective antagonists of such an interaction.
Here, we describe the protein crystal structure guided optimization
of prototypic compound <b>2</b> (<i>K</i><sub>dis</sub> = 7 μM), leading to identification of more potent antagonist <b>47</b> (<i>K</i><sub>dis</sub> = 0.3 μM)
Discovery of Potent and Selective Allosteric Inhibitors of Protein Arginine Methyltransferase 3 (PRMT3)
PRMT3 catalyzes the asymmetric dimethylation
of arginine residues
of various proteins. It is crucial for maturation of ribosomes and
has been implicated in several diseases. We recently disclosed a highly
potent, selective, and cell-active allosteric inhibitor of PRMT3,
compound <b>4</b>. Here, we report comprehensive structure–activity
relationship studies that target the allosteric binding site of PRMT3.
We conducted design, synthesis, and evaluation of novel compounds
in biochemical, selectivity, and cellular assays that culminated in
the discovery of <b>4</b> and other highly potent (IC<sub>50</sub> values: ∼10–36 nM), selective, and cell-active allosteric
inhibitors of PRMT3 (compounds <b>29</b>, <b>30</b>, <b>36</b>, and <b>37</b>). In addition, we generated compounds
that are very close analogs of these potent inhibitors but displayed
drastically reduced potency as negative controls (compounds <b>49</b>–<b>51</b>). These inhibitors and negative
controls are valuable chemical tools for the biomedical community
to further investigate biological functions and disease associations
of PRMT3