Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer.

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.

A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells.

Lipids play central roles in physiology and disease, where their structural, metabolic, and signaling functions often arise from interactions with proteins. Here, we describe a set of lipid-based chemical proteomic probes and their global interaction map in mammalian cells. These interactions involve hundreds of proteins from diverse functional classes and frequently occur at sites of drug action. We determine the target profiles for several drugs across the lipid-interaction proteome, revealing that its ligandable content extends far beyond traditionally defined categories of druggable proteins. In further support of this finding, we describe a selective ligand for the lipid-binding protein nucleobindin-1 (NUCB1) and show that this compound perturbs the hydrolytic and oxidative metabolism of endocannabinoids in cells. The described chemical proteomic platform thus provides an integrated path to both discover and pharmacologically characterize a wide range of proteins that participate in lipid pathways in cells.

Modification of residue 42 of the active site loop with a lysine-mimetic side chain rescues isochorismate-pyruvate lyase activity in Pseudomonas aeruginosa PchB.

PchB is an isochorismate-pyruvate lyase from Pseudomonas aeruginosa. A positively charged lysine residue is located in a flexible loop that behaves as a lid to the active site, and the lysine residue is required for efficient production of salicylate. A variant of PchB that lacks the lysine at residue 42 has a reduced catalytic free energy of activation of up to 4.4 kcal/mol. Construction of a lysine isosteric residue bearing a positive charge at the appropriate position leads to the recovery of 2.5-2.7 kcal/mol (about 60%) of the 4.4 kcal/mol by chemical rescue. Exogenous addition of ethylamine to the K42A variant leads to a neglible recovery of activity (0.180 kcal/mol, roughly 7% rescue), whereas addition of propylamine caused an additional modest loss in catalytic power (0.056 kcal/mol, or 2% loss). This is consistent with the view that (a) the lysine-42 residue is required in a specific conformation to stabilize the transition state and (b) the correct conformation is achieved for a lysine-mimetic side chain at site 42 in the course of loop closure, as expected for transition-state stabilization by the side chain ammonio function. That the positive charge is the main effector of transition state stabilization is shown by the construction of a lysine-isosteric residue capable of exerting steric effects and hydrogen bonding but not electrostatic effects, leading to a modest increase of catalytic power (0.267-0.505 kcal/mol of catalytic free energy, or roughly 6-11% rescue).

Two structures of an N-hydroxylating flavoprotein monooxygenase: ornithine hydroxylase from Pseudomonas aeruginosa.

The ornithine hydroxylase from Pseudomonas aeruginosa (PvdA) catalyzes the FAD-dependent hydroxylation of the side chain amine of ornithine, which is subsequently formylated to generate the iron-chelating hydroxamates of the siderophore pyoverdin. PvdA belongs to the class B flavoprotein monooxygenases, which catalyze the oxidation of substrates using NADPH as the electron donor and molecular oxygen. Class B enzymes include the well studied flavin-containing monooxygenases and Baeyer-Villiger monooxygenases. The first two structures of a class B N-hydroxylating monooxygenase were determined with FAD in oxidized (1.9 Å resolution) and reduced (3.03 Å resolution) states. PvdA has the two expected Rossmann-like dinucleotide-binding domains for FAD and NADPH and also a substrate-binding domain, with the active site at the interface between the three domains. The structures have NADP(H) and (hydroxy)ornithine bound in a solvent-exposed active site, providing structural evidence for substrate and co-substrate specificity and the inability of PvdA to bind FAD tightly. Structural and biochemical evidence indicates that NADP(+) remains bound throughout the oxidative half-reaction, which is proposed to shelter the flavin intermediates from solvent and thereby prevent uncoupling of NADPH oxidation from hydroxylated product formation.

pH Dependence of catalysis by Pseudomonas aeruginosa isochorismate-pyruvate lyase: implications for transition state stabilization and the role of lysine 42.

An isochorismate-pyruvate lyase with adventitious chorismate mutase activity from Pseudomonas aerugionsa (PchB) achieves catalysis of both pericyclic reactions in part by the stabilization of reactive conformations and in part by electrostatic transition-state stabilization. When the active site loop Lys42 is mutated to histidine, the enzyme develops a pH dependence corresponding to a loss of catalytic power upon deprotonation of the histidine. Structural data indicate that the change is not due to changes in active site architecture, but due to the difference in charge at this key site. With loss of the positive charge on the K42H side chain at high pH, the enzyme retains lyase activity at ∼100-fold lowered catalytic efficiency but loses detectable mutase activity. We propose that both substrate organization and electrostatic transition state stabilization contribute to catalysis. However, the dominant reaction path for catalysis is dependent on reaction conditions, which influence the electrostatic properties of the enzyme active site amino acid side chains.

Mechanistic and structural studies of the N-hydroxylating flavoprotein monooxygenases.

The N-hydroxylating flavoprotein monooxygenases are siderophore biosynthetic enzymes that catalyze the hydroxylation of the sidechain amino-group of ornithine or lysine or the primary amino-group of putrescine. This hydroxylated product is subsequently formylated or acylated and incorporated into the siderophore. Importantly, the modified amino-group is a hydroxamate and serves as an iron chelating moiety in the siderophore. This review describes recent work to characterize the ornithine hydroxylases from Pseudomonas aeruginosa (PvdA) and Aspergillus fumigatus (SidA) and the lysine hydroxylase from Escherichia coli (IucD). This includes summaries of steady and transient state kinetic data for all three enzymes and the X-ray crystallographic structure of PvdA.

Structure-function analyses of isochorismate-pyruvate lyase from Pseudomonas aeruginosa suggest differing catalytic mechanisms for the two pericyclic reactions of this bifunctional enzyme.

The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) catalyzes two pericyclic reactions in a single active site. PchB physiologically produces salicylate and pyruvate from isochorismate for ultimate incorporation of the salicylate into the siderophore pyochelin. PchB also produces prephenate from chorismate, most likely due to structural homology to the Escherchia coli chorismate mutase. The molecular basis of catalysis among enzymatic pericyclic reactions is a matter of debate, one view holding that catalysis may be derived from electrostatic transition state stabilization and the opposing view that catalysis is derived from the generation of a reactive substrate conformation. Mutant forms of PchB were generated by site-directed mutagenesis at the site (K42) hypothesized to be key for electrostatic transition state stabilization (K42A, K42Q, K42E, and K42H). The loop containing K42 is mobile, and a mutant to slow loop dynamics was also designed (A43P). Finally, a previously characterized mutation (I87T) was also produced. Circular dichroism was used to assess the overall effect on secondary structure as a result of the mutations, and X-ray crystallographic structures are reported for K42A with salicylate and pyruvate bound and for apo-I87T. The data illustrate that the active site architecture is maintained in K42A-PchB, which indicates that differences in activity are not caused by secondary structural changes or by differences in active site loop conformation but rather by the chemical nature of this key residue. In contrast, the I87T structure demonstrates considerable mobility, suggesting that loop dynamics and conformational plasticity may be important for efficient catalysis. Finally, the mutational effects on k(cat) provide evidence that the two activities of PchB are not covariant and that a single hypothesis may not provide a sufficient explanation for catalysis.