Mini review

Signal transduction pathways involving RAS

Here, we discuss the principles of allosteric activating mutations, propagation downstream of the signals that they prompt, and allosteric drugs, with examples from the Ras signaling network. We focus on Abl kinase where mutations shift the landscape toward the active, imatinib binding-incompetent conformation, likely resulting in the high affinity ATP outcompeting drug binding. Recent pharmacological innovation extends to allosteric inhibitor (GNF-5)-linked PROTAC, targeting Bcr-Abl1 myristoylation site, and broadly, allosteric heterobifunctional degraders that destroy targets, rather than inhibiting them. Designed chemical linkers in bifunctional degraders can connect the allosteric ligand that binds the target protein and the E3 ubiquitin ligase warhead anchor. The physical properties and favored conformational state of the engineered linker can precisely coordinate the distance and orientation between the target and the recruited E3. Allosteric PROTACs, noncompetitive molecular glues, and bitopic ligands, with covalent links of allosteric ligands and orthosteric warheads, increase the effective local concentration of productively oriented and placed ligands. Through covalent chemical or peptide linkers, allosteric drugs can collaborate with competitive drugs, degrader anchors, or other molecules of choice, driving innovative drug discovery.

Intuitively, functional states should be targeted; not nonfunctional ones. So why could drugging the inactive K-Ras4B^G12C work—but drugging the inactive kinase will likely not? The reason is the distinct oncogenic mechanisms. Kinase driver mutations work by stabilizing the active state and/or destabilizing the inactive state. Either way, oncogenic kinases are mostly in the active state. Ras driver mutations work by quelling its deactivation mechanisms, GTP hydrolysis, and nucleotide exchange. Covalent inhibitors that bind to the inactive GDP-bound K-Ras4B^G12C conformation can thus work. By contrast, in kinases, allosteric inhibitors work by altering the active-site conformation to favor orthosteric drugs. From the translational standpoint this distinction is vital: it expedites effective pharmaceutical development and extends the drug classification based on the mechanism of action. Collectively, here we postulate that drug action relates to blocking the mechanism of activation, not to whether the protein is in the active or inactive state.

There has been growing interest in applying the empirical valence bond approach to a range of (bio)chemical problems, primarily to study enzymatic and non-enzymatic catalysis, but also to studying other processes such as excited state chemistry and reaction dynamics. Despite its apparent theoretical simplicity, this approach is a powerful computational tool that can be used to reproduce and rationalize a wide range of experimental observables, such as linear free energy relationships, kinetic isotope effects, and temperature effects on reaction rates. We provide here both a theoretical background for this approach, as well as highlighting several of its broad applications in computational physical organic chemistry.

Pseudomonas aeruginosa is a Gram-negative bacterium that grows in diverse environments, including soil, water, and numerous sites of human infection. P. aeruginosa is a nosocomial pathogen for patients with preexisting lung disease, such as cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD), or who undergo medical procedures that interfere with the normal innate barrier defenses, such as mechanical ventilation or surgery. P. aeruginosa utilizes exotoxin A and four type III secreted effectors as major virulence factors. This chapter discusses the structural, molecular, and functional properties of these toxins with respect to P. aeruginosa pathogenesis and as candidates for vaccine development toward the control of infection.

Tight junctions (TJs) and adherens junctions (AJs) are dynamic structures linked to the actin cytoskeleton, which control the paracellular permeability of epithelial and endothelial barriers. TJs and AJs are strictly regulated in a spatio-temporal manner by a complex signaling network, including Rho/Ras-GTPases, which have a pivotal role. Rho preferentially regulates TJs by controlling the contraction of apical acto-myosin filaments, whereas Rac/Cdc42 mainly coordinate the assembly–disassembly of AJ components. However, a subtle balance of Rho/Ras-GTPase activity and interplay between these molecules is required to maintain an optimal organization and function of TJs and AJs. Conversely, integrity of intercellular junctions generates signals through Rho-GTPases, which are involved in the regulation of multiple cellular processes. Rho/Ras-GTPases and the control of intercellular junctions are the target of various bacterial toxins responsible for severe diseases in man and animals, and are part of their mechanism of action. This review focuses on the regulation of TJs and AJs by Rho/Ras-GTPases through molecular approaches and bacterial toxins.

In the Ras·RasGAP complex, hydrolysis of guanosine triphosphate is strongly accelerated GAP as compared to Ras alone. This is largely attributed to the arginine finger R789^GAP pointing to AlF_x in the transition state analogue. We performed QM/MM simulations where triphosphate was treated using the quantum mechanical method of density functional theory, while the protein complex and water environment were described classically using MD. Compared to Ras, the crucial electron shift, bond stretching and distortion towards an eclipsed γ-to-β orientation are much more pronounced. The arginine finger is shown to act by displacing water out of the binding niche. The resulting enhanced electrostatic field catalyses the cleavage step.