Targeting N-Myc in neuroblastoma with selective Aurora kinase A degraders.

The N-Myc transcription factor, encoded by MYCN, is a mechanistically validated, yet challenging, target for neuroblastoma (NB) therapy development. In normal neuronal progenitors, N-Myc undergoes rapid degradation, while, in MYCN-amplified NB cells, Aurora kinase A (Aurora-A) binds to and stabilizes N-Myc, resulting in elevated protein levels. Here, we demonstrate that targeted protein degradation of Aurora-A decreases N-Myc levels.

Exploring the conformational landscapes of protein kinases: perspectives from FRET and DEER.

Conformational changes of catalytically-important structural elements are a key feature of the regulation mechanisms of protein kinases and are important for dictating inhibitor binding modes and affinities. The lack of widely applicable methods for tracking kinase conformational changes in solution has hindered our understanding of kinase regulation and our ability to design conformationally selective inhibitors. Here we provide an overview of two recently developed methods that detect conformational changes of the regulatory activation loop and αC-helix of kinases and that yield complementary information about allosteric mechanisms.

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors.

Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding.

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception.

Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors.

Screening through Lead Optimization of High Affinity, Allosteric Cyclin-Dependent Kinase 2 (CDK2) Inhibitors as Male Contraceptives That Reduce Sperm Counts in Mice.

Although cyclin-dependent kinase 2 (CDK2) is a validated target for both cancer and contraception, developing a CDK2 inhibitor with exquisite selectivity has been challenging due to the structural similarity of the ATP-binding site, where most kinase inhibitors bind. We previously discovered an allosteric pocket in CDK2 with the potential to bind a selective compound and then discovered and structurally confirmed an anthranilic acid scaffold that binds this pocket with high affinity. These allosteric inhibitors are selective for CDK2 over structurally similar CDK1 and show contraceptive potential.

Allostery governs Cdk2 activation and differential recognition of CDK inhibitors.

Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding.

Cooperativity Between Orthosteric Inhibitors and Allosteric Inhibitor 8-Anilino-1-Naphthalene Sulfonic Acid (ANS) in Cyclin-Dependent Kinase 2.

While kinases have been attractive targets to combat many diseases, including cancer, selective kinase inhibition has been challenging, because of the high degree of structural homology in the active site, where many kinase inhibitors bind. We have previously discovered that 8-anilino-1-naphthalene sulfonic acid (ANS) binds an allosteric pocket in cyclin-dependent kinase 2 (Cdk2). Here, we detail the positive cooperativity between ANS and orthosteric Cdk2 inhibitors dinaciclib and roscovitine, which increase the affinity of ANS toward Cdk2 5-fold to 10-fold, and the relatively noncooperative effects of ATP.

The pseudokinase domains of guanylyl cyclase-A and -B allosterically increase the affinity of their catalytic domains for substrate.

Natriuretic peptides regulate multiple physiologic systems by activating transmembrane receptors containing intracellular guanylyl cyclase domains, such as GC-A and GC-B, also known as Npr1 and Npr2, respectively. Both enzymes contain an intracellular, phosphorylated pseudokinase domain (PKD) critical for activation of the C-terminal cGMP-synthesizing guanylyl cyclase domain. Because ATP allosterically activates GC-A and GC-B, we investigated how ATP binding to the PKD influenced guanylyl cyclase activity.

Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states.

Protein kinases undergo large-scale structural changes that tightly regulate function and control recognition by small-molecule inhibitors. Methods for quantifying the conformational effects of inhibitors and linking them to an understanding of selectivity patterns have long been elusive. We have developed an ultrafast time-resolved fluorescence methodology that tracks structural movements of the kinase activation loop in solution with angstrom-level precision, and can resolve multiple structural states and quantify conformational shifts between states.

An Open Library of Human Kinase Domain Constructs for Automated Bacterial Expression.

Kinases play a critical role in cellular signaling and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Therapeutics targeting kinases currently account for roughly 50% of cancer drug discovery efforts. The ability to explore human kinase biochemistry and biophysics in the laboratory is essential to designing selective inhibitors and studying drug resistance.

The multifaceted allosteric regulation of Aurora kinase A.

The protein kinase Aurora A (AurA) is essential for the formation of bipolar mitotic spindles in all eukaryotic organisms. During spindle assembly, AurA is activated through two different pathways operating at centrosomes and on spindle microtubules. Recent studies have revealed that these pathways operate quite differently at the molecular level, activating AurA through multifaceted changes to the structure and dynamics of the kinase domain.

A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation.

Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100 fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift from the DFG-Out to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation triggers a switch within the DFG-In subpopulation from an autoinhibited DFG-In substate to an active DFG-In substate, leading to catalytic activation.

A water-mediated allosteric network governs activation of Aurora kinase A.

The catalytic activity of many protein kinases is controlled by conformational changes of a conserved Asp-Phe-Gly (DFG) motif. We used an infrared probe to track the DFG motif of the mitotic kinase Aurora A (AurA) and found that allosteric activation by the spindle-associated protein Tpx2 involves an equilibrium shift toward the active DFG-in state. Förster resonance energy transfer experiments show that the activation loop undergoes a nanometer-scale movement that is tightly coupled to the DFG equilibrium.

A conserved water-mediated hydrogen bond network defines bosutinib's kinase selectivity.

Kinase inhibitors are important cancer drugs, but they tend to display limited target specificity, and their target profiles are often challenging to rationalize in terms of molecular mechanism. Here we report that the clinical kinase inhibitor bosutinib recognizes its kinase targets by engaging a pair of conserved structured water molecules in the active site and that many other kinase inhibitors share a similar recognition mechanism. Using the nitrile group of bosutinib as an infrared probe, we show that the gatekeeper residue and one other position in the ATP-binding site control access of the drug to the structured water molecules and that the amino acids found at these positions account for the kinome-wide target spectrum of the drug.

Experimental quantification of electrostatics in X-H···π hydrogen bonds.

Hydrogen bonds are ubiquitous in chemistry and biology. The physical forces that govern hydrogen-bonding interactions have been heavily debated, with much of the discussion focused on the relative contributions of electrostatic vs quantum mechanical effects. In principle, the vibrational Stark effect, the response of a vibrational mode to electric field, can provide an experimental method for parsing such interactions into their electrostatic and nonelectrostatic components.

Structural and spectroscopic analysis of the kinase inhibitor bosutinib and an isomer of bosutinib binding to the Abl tyrosine kinase domain.

Chronic myeloid leukemia (CML) is caused by the kinase activity of the BCR-Abl fusion protein. The Abl inhibitors imatinib, nilotinib and dasatinib are currently used to treat CML, but resistance to these inhibitors is a significant clinical problem. The kinase inhibitor bosutinib has shown efficacy in clinical trials for imatinib-resistant CML, but its binding mode is unknown.

Solvent-induced infrared frequency shifts in aromatic nitriles are quantitatively described by the vibrational Stark effect.

The physical properties of solvents strongly affect the spectra of dissolved solutes, and this phenomenon can be exploited to gain insight into the solvent-solute interaction. The large solvatochromic shifts observed for many dye molecules in polar solvents are due to variations in the solvent reaction field, and these shifts are widely used to estimate the change in the dye's dipole moment upon photoexcitation, which is typically on the order of ∼1-10 D. In contrast, the change in dipole moment for vibrational transitions is approximately 2 orders of magnitude smaller.

Direct measurements of electric fields in weak OH···π hydrogen bonds.

Hydrogen bonds and aromatic interactions are of widespread importance in chemistry, biology, and materials science. Electrostatics play a fundamental role in these interactions, but the magnitude of the electric fields that support them has not been quantified experimentally. Phenol forms a weak hydrogen bond complex with the π-cloud of benzene, and we used this as a model system to study the role of electric fields in weak OH···π hydrogen bonds.

Phosphate vibrations probe local electric fields and hydration in biomolecules.

The role of electric fields in important biological processes such as binding and catalysis has been studied almost exclusively by computational methods. Experimental measurements of the local electric field in macromolecules are possible using suitably calibrated vibrational probes. Here we demonstrate that the vibrational transitions of phosphate groups are highly sensitive to an electric field and show how that sensitivity can be quantified, allowing electric field measurements to be made in phosphate-containing biological systems without chemical modification.

The tyrosine kinase Csk dimerizes through Its SH3 domain.

The Src family kinases possess two sites of tyrosine phosphorylation that are critical to the regulation of kinase activity. Autophosphorylation on an activation loop tyrosine residue (Tyr 416 in commonly used chicken c-Src numbering) increases catalytic activity, while phosphorylation of a C-terminal tyrosine (Tyr 527 in c-Src) inhibits activity. The latter modification is achieved by the tyrosine kinase Csk (C-terminal Src Kinase), but the complete inactivation of the Src family kinases also requires the dephosphorylation of the activation loop tyrosine.

Structural basis for the recognition of c-Src by its inactivator Csk.

The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src Kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. We have determined the crystal structure of a complex between the kinase domains of Csk and c-Src at 2.

A Src-like inactive conformation in the abl tyrosine kinase domain.

The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML). The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG) motif is flipped by approximately 180 degrees with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src.