A precision oncology approach to the pharmacological targeting of mechanistic dependencies in neuroendocrine tumors.

We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology.

Making structural sense of dimerization interfaces of delta opioid receptor homodimers.

Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed "4" dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed "4/5" dimer) in an explicit lipid-water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.

Recent applications of Kirkwood-Buff theory to biological systems.

The effect of cosolvents on biomolecular equilibria has traditionally been rationalized using simple binding models. More recently, a renewed interest in the use of Kirkwood-Buff (KB) theory to analyze solution mixtures has provided new information on the effects of osmolytes and denaturants and their interactions with biomolecules. Here we review the status of KB theory as applied to biological systems. In particular, the existing models of denaturation are analyzed in terms of KB theory, and the use of KB theory to interpret computer simulation data for these systems is discussed.

NMR structure and dynamic studies of an anion-binding, channel-forming heptapeptide.

The synthetic peptide (C(18)H(37))(2)NCOCH(2)OCH(2)CON-(Gly)(3)-Pro-(Gly)(3)-OCH(2)Ph forms chloride-selective channels in liposomes and exhibits voltage-gating properties in planar phospholipid bilayers. The peptide fragment of the channel is based on a conserved motif in naturally occurring chloride transporters. Membrane-anchoring residues at the N- and C-terminal ends augment the peptide. NMR spectra (1D and 2D) of the channel in CDCl(3) showed significant variation in the absence and presence of stoichiometric tetrabutylammonium chloride (Bu(4)NCl). One-dimensional solution-state NMR titration studies combined with computational molecular simulation studies indicate that the peptide interacts with the salt as an ion pair and H-bonds chloride. To our knowledge, this is the first structural analysis of any synthetic anion-channel salt complex.

Modeling and simulation of the human delta opioid receptor.

A model for the human delta opioid receptor has been generated via sequence alignment, structure building using the crystal structure of bovine rhodopsin as a template, and refinement by molecular dynamics simulation. The model building suggested that, in addition to the previously postulated interaction between D128 and Y308, an internal salt bridge also exists between residues D128 and R192, both of which are conserved in all the opioid receptors. The model and salt bridge were then shown to be stable during a 20-nsec simulation in a lipid bilayer. It is therefore proposed that both of these interactions play a role in stabilizing the inactive state of the receptor. The model is also used in an effort to rationalize many of the mutational studies performed on delta opioid receptors, and to suggest a plausible explanation for the differences between known delta opioid agonists and antagonists.

A conformational analysis of leucine enkephalin as a function of pH.

The conformations of Leu enkephalin in aqueous solution have been investigated as a function of pH using molecular dynamics simulations. The simulations suggest the peptide backbone exists as a mixture of folded and unfolded forms (approximately 50% each) at neutral pH, but is always unfolded at low or high pH. The folded form at neutral pH possesses a 2 --> 5 hydrogen bond and a close head to tail separation. No significant intramolecular hydrogen bonding of the carbonyl oxygens was observed in either the folded or unfolded forms of the peptide. Analysis of the Gly carbonyl oxygens and terminal groups indicated that, while the conformational population distribution of Leu enkephalin did vary noticeably as a function of pH, their hydration was essentially independent of pH and in agreement with the available NMR data. Further study indicated that the unfolded state of the peptide was not random in nature and consisted of one major unfolded backbone arrangement stabilized by a persistent hydrophobic interaction between the side chains of Tyr and Leu.