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
The cyclin D1-cyclin-dependent kinase 4 (CDK4) complex is a key regulator of the transition through the G₁ phase of the cell cycle. Among the cyclin/CDKs, CDK4 and cyclin D1 are the most frequently ...activated by somatic genetic alterations in multiple tumor types. Thus, aberrant regulation of the CDK4/cyclin D1 pathway plays an essential role in oncogenesis; hence, CDK4 is a genetically validated therapeutic target. Although X-ray crystallographic structures have been determined for various CDK/cyclin complexes, CDK4/cyclin D1 has remained highly refractory to structure determination. Here, we report the crystal structure of CDK4 in complex with cyclin D1 at a resolution of 2.3 Å. Although CDK4 is bound to cyclin D1 and has a phosphorylated T-loop, CDK4 is in an inactive conformation and the conformation of the heterodimer diverges from the previously known CDK/cyclin binary complexes, which suggests a unique mechanism for the process of CDK4 regulation and activation.
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 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.
In-gel activity-based protein profiling (ABPP) offers rapid assessment of the proteome-wide selectivity and target engagement of a chemical tool. Here we demonstrate the use of the inverse electron ...demand Diels Alder (IEDDA) click reaction for in-gel ABPP by evaluating the selectivity profile and target engagement of a covalent ERK1/2 probe tagged with a trans-cyclooctene group. The chemical probe was shown to bind covalently to Cys166 of ERK2 using protein MS and X-ray crystallography, and displayed submicromolar GI50s in A375 and HCT116 cells. In both cell lines, the probe demonstrated target engagement and a good selectivity profile at low concentrations, which was lost at higher concentrations. The IEDDA cycloaddition enabled fast and quantitative fluorescent tagging for readout with a high background-to-noise ratio and thereby provides a promising alternative to the commonly used copper catalysed alkyne-azide cycloaddition.
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.
In-gel activity-based protein profiling (ABPP) offers rapid assessment of the proteome-wide selectivity and target engagement of a chemical tool. Here we demonstrate the use of the inverse electron ...demand Diels Alder (IEDDA) click reaction for in-gel ABPP by evaluating the selectivity profile and target engagement of a covalent ERK1/2 probe tagged with a
trans
-cyclooctene group. The chemical probe was shown to bind covalently to Cys166 of ERK2 using protein MS and X-ray crystallography, and displayed submicromolar GI
50
s in A375 and HCT116 cells. In both cell lines, the probe demonstrated target engagement and a good selectivity profile at low concentrations, which was lost at higher concentrations. The IEDDA cycloaddition enabled fast and quantitative fluorescent tagging for readout with a high background-to-noise ratio and thereby provides a promising alternative to the commonly used copper catalysed alkyne-azide cycloaddition.
The
trans
-cyclooctenol/tetrazine click reaction was used for in-gel ABPP to determine the proteome-wide selectivity profile of a covalent ERK1/2 inhibitor.
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