Cancer cells exhibit several unique metabolic phenotypes that are critical for cell growth and proliferation. Specifically, they overexpress the M2 isoform of the tightly regulated enzyme pyruvate ...kinase (PKM2), which controls glycolytic flux, and are highly dependent on de novo biosynthesis of serine and glycine. Here we describe a new rheostat-like mechanistic relationship between PKM2 activity and serine biosynthesis. We show that serine can bind to and activate human PKM2, and that PKM2 activity in cells is reduced in response to serine deprivation. This reduction in PKM2 activity shifts cells to a fuel-efficient mode in which more pyruvate is diverted to the mitochondria and more glucose-derived carbon is channelled into serine biosynthesis to support cell proliferation.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
KEAP1 is the key regulator of the NRF2-mediated cytoprotective response, and increasingly recognized as a target for diseases involving oxidative stress. Pharmacological intervention has focused on ...molecules that decrease NRF2-ubiquitination through covalent modification of KEAP1 cysteine residues, but such electrophilic compounds lack selectivity and may be associated with off-target toxicity. We report here the first use of a fragment-based approach to directly target the KEAP1 Kelch–NRF2 interaction. X-ray crystallographic screening identified three distinct “hot-spots” for fragment binding within the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment hit to molecules with nanomolar affinity for KEAP1 while maintaining drug-like properties. This work resulted in a promising lead compound which exhibits tight and selective binding to KEAP1, and activates the NRF2 antioxidant response in cellular and in vivo models, thereby providing a high quality chemical probe to explore the therapeutic potential of disrupting the KEAP1–NRF2 interaction.
Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) are currently generating significant interest in clinical development as potential treatments for cancer. In a preceding ...publication (DOI: 10.1021/jm100059d) we describe Astex’s approach to screening fragments against Hsp90 and the subsequent optimization of two hits into leads with inhibitory activities in the low nanomolar range. This paper describes the structure guided optimization of the 2,4-dihydroxybenzamide lead molecule 1 and details some of the drug discovery strategies employed in the identification of AT13387 (35), which has progressed through preclinical development and is currently being tested in man.
Higher throughput thermodynamic measurements can provide value in structure-based drug discovery during fragment screening, hit validation, and lead optimization. Enthalpy can be used to detect and ...characterize ligand binding, and changes that affect the interaction of protein and ligand can sometimes be detected more readily from changes in the enthalpy of binding than from the corresponding free-energy changes or from protein–ligand structures. Newer, higher throughput calorimeters are being incorporated into the drug discovery process. Improvements in titration calorimeters come from extensions of a mature technology and face limitations in scaling. Conversely, array calorimetry, an emerging technology, shows promise for substantial improvements in throughput and material utilization, but improved sensitivity is needed.
The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several ...hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET.
The KEAP1–NRF2-mediated cytoprotective response plays a key role in cellular homoeostasis. Insufficient NRF2 signaling during chronic oxidative stress may be associated with the pathophysiology of ...several diseases with an inflammatory component, and pathway activation through direct modulation of the KEAP1–NRF2 protein–protein interaction is being increasingly explored as a potential therapeutic strategy. Nevertheless, the physicochemical nature of the KEAP1–NRF2 interface suggests that achieving high affinity for a cell-penetrant druglike inhibitor might be challenging. We recently reported the discovery of a highly potent tool compound which was used to probe the biology associated with directly disrupting the interaction of NRF2 with the KEAP1 Kelch domain. We now present a detailed account of the medicinal chemistry campaign leading to this molecule, which included exploration and optimization of protein–ligand interactions in three energetic “hot spots” identified by fragment screening. In particular, we also discuss how consideration of ligand conformational stabilization was important to its development and present evidence for preorganization of the lead compound which may contribute to its high affinity and cellular activity.
β-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The ...identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.β-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.
The various oligomeric states of the M2 isoform of pyruvate kinase (PKM2) were distinguished using native mass spectrometry. The effect of PKM2 concentration on its dimer–tetramer equilibrium was ...monitored, and a value for the dissociation constant (K d) of the two species was estimated to be 0.95 μM. Results of binding of fructose-1,6-bisphosphate (FBP) to PKM2 are shown and provide insight into the allosteric mechanism and changes in the oligomerization status of PKM2. The average K d for binding of FBP to the PKM2 tetramer was estimated to be 7.5 μM. It is concluded that four molecules of FBP bind to the active PKM2 tetramer whereas binding of FBP to the PKM2 dimer was not observed. It is suggested that either FBP potentiates rapid tetramer formation after binding to apo PKM2 dimers or FBP binds to PKM2 apo tetramers, thus driving the dimer–tetramer equilibrium in the direction of fully FBP-bound tetramer. The binding occurs in a highly positively cooperative manner with a Hill coefficient (n) of 3.
Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to ...identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.
Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets ...for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (
) through structure-guided optimization of our previously published isoindolinone lead (
). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.
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