The Future of (Radio)-Labeled Compounds in Research and Development within the Life Science Industry.

In this review the applications of isotopically labeled compounds are discussed and put into the context of their future impact in the life sciences. Especially discussing their use in the pharma and crop science industries to follow their fate in the environment, in vivo or in complex matrices to understand the potential harm of new chemical structures and to increase the safety of human society.

Morphological Profiling Identifies a Common Mode of Action for Small Molecules with Different Targets.

Unbiased morphological profiling of bioactivity, for example, in the cell painting assay (CPA), enables the identification of a small molecule's mode of action based on its similarity to the bioactivity of reference compounds, irrespective of the biological target or chemical similarity. This is particularly important for small molecules with nonprotein targets as these are rather difficult to identify with widely employed target-identification methods. We employed morphological profiling using the CPA to identify compounds that are biosimilar to the iron chelator deferoxamine. Structurally different compounds with different annotated cellular targets provoked a shared physiological response, thereby defining a cluster based on their morphological fingerprints. This cluster is based on a shared mode of action and not on a shared target, that is, cell-cycle modulation in the S or G2 phase. Hierarchical clustering of morphological fingerprints revealed subclusters that are based on the mechanism of action and could be used to predict target-related bioactivity.

Flat corannulene: when a transition state becomes a stable molecule.

Flat corannulene has been considered so far only as a transition state of the bowl-to-bowl inversion process. This study was driven by the prediction that substituents with strong steric repulsion could destabilize the bowl-shaped conformation of this molecule to such an extent that the highly unstable planar geometry would become an isolable molecule. To examine the substituents' effect on the corannulene bowl depth, optimized structures for the highly-congested decakis(t-butylsulfido)corannulene were calculated. The computations, performed with both the M06-2X/def2-TZVP and the B3LYP/def2-TZVP methods (the latter with and without Grimme's D3 dispersion correction), predict that this molecule can achieve two minimum structures: a flat carbon framework and a bowl-shaped structure, which are very close in energy. This rather unusual compound was easily synthesized from decachlorocorannulene under mild reaction conditions, and X-ray crystallographic studies gave similar results to the theoretical predictions. This compound crystallized in two different polymorphs, one exhibiting a completely flat corannulene core and the other having a bowl-shaped conformation.

Identification of Quinolinols as Activators of TEAD-Dependent Transcription.

The transcriptional co-regulators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are the vertebrate downstream effectors of the Hippo signaling pathway that controls various physiological and pathological processes. YAP and TAZ pair with the TEAD (TEA domain) family of transcription factors to initiate transcription. We previously identified a tractable pocket in TEADs, which has been physiologically shown to bind palmitate. Herein, a TEAD-palmitate interaction screen was developed to select small molecules occupying the palmitate-binding pocket (PBP) of TEADs. We show that quinolinols were TEAD-binding compounds that augment YAP/TAZ-TEAD activity, which was verified using TEAD reporter assay, RT-qPCR, and RNA-Seq analyses. Structure-activity relationship investigations uncovered the quinolinol substituents that are necessary for TEAD activation. We reveal a novel mechanism where quinolinols stabilize YAP/TAZ protein levels by occupying the PBP. The enhancement of YAP activity by quinolinols accelerates the in vivo wound closure in a mouse wound-healing model. Although small molecules that occupy the PBP have been shown to inhibit YAP/TAZ-TEAD activity, leveraging PBP to activate TEADs is a novel approach.

Small-Molecule Inhibition of the UNC-Src Interaction Impairs Dynamic Src Localization in Cells.

Interference with the signaling activity of the N-myristoylated nonreceptor protein tyrosine kinase Src is considered a viable approach in anti-cancer drug discovery. However, ATP-competitive Src inhibitors have not reached the clinic yet and alternative approaches are in high demand. The UNC119A/B proteins bind the myristoylated N terminus of Src and thereby mediate energy-driven spatial cycles that maintain Src enrichment at the plasma membrane, which is critical for Src signaling activity. We describe the discovery of a potent and specific inhibitor of the UNC119-Src interaction with unprecedented chemotype. The inhibitor binds to UNC119 in cells, and induces redistribution of Src to endomembranes and reduction of activating Src autophosphorylation on Y419. UNC119 inhibition in Src-dependent colorectal cancer cells results in the specific reduction of cell growth and clonogenic potential. Our results demonstrate that small-molecule interference with the dynamics of the Src spatial cycle may provide an opportunity to impair oncogenic Src signaling.

Photoactivatable Myristic Acid Probes for UNC119-Cargo Interactions.

Protein myristoylation plays key roles in biological processes, for instance, in membrane attachment and activation of proteins and in mediating protein-protein and protein-lipid interactions. Furthermore, myristoylated proteins are involved in disorders, including cancer and viral infections. Therefore, new tools to study protein myristoylation are in high demand. Herein, we report the development of photoactivatable probes, based on a diazirine-substituted analogue of myristic acid. The probes bind to and, upon irradiation, covalently label the lipid-binding chaperone protein uncoordinated 119 (UNC119). UNC119 increases overall solubility and regulates specifically the transport of myristoylated proteins between intercellular membranes. The binding mode of the probes is similar to that of the myristate moiety, and the residues inside the hydrophobic pocket of UNC119 proteins that are critical for covalent binding have been identified. The interaction with UNC119 was also demonstrated in cell lysate by means of affinity enrichment. Moreover, it is shown that the myristate analogue can be incorporated into peptide substrates by N-myristoyl transferases of Leishmania and Trypanosoma protozoan parasites.

UNC119A Decreases the Membrane Binding of Myristoylated c-Src.

Plasma membrane localization of myristoylated c-Src, a proto-oncogene protein-tyrosine kinase, is required for its signaling activity. Recent studies proposed that UNC119 protein functions as a solubilizing factor for myristoylated proteins, thereby regulating their subcellular distribution and signaling. The underlying molecular mechanism by which UNC119 regulates the membrane binding of c-Src has remained elusive. By combining different biophysical techniques, we have found that binding of a myristoylated c-Src-derived N-terminal peptide (Myr-Src) by UNC119A results in a reduced membrane binding affinity of the peptide, due to the competition of binding to membranes. The dissociation of Myr-Src from membranes is facilitated in the presence of UNC119A, as a consequence of which the clustering propensity of this peptide on the membrane is partially impaired. By these means, UNC119A is able to regulate c-Src spatially in the cytoplasm and on cellular membranes, and this has important implications for its cellular signaling.

Lateral Organization of Host Heterogeneous Raft-like Membranes Altered by the Myristoyl Modification of Tyrosine Kinase c-Src.

Membrane-bound c-Src non-receptor tyrosine kinase, unlike other acyl-modified lipid-anchored proteins, anchors to the membrane by a myristoyl chain along with a polybasic residue stretch, which is shorter in chain length than its host membrane. The packing defect arising from this mismatched chain length of the host and the lipid anchor significantly affects the lateral organization of heterogeneous membranes. We reveal the mixing of phase domains and formation of novel nanoscale-clusters upon membrane binding of the Myr-Src (2-9) peptide. Fluorescence cross correlation spectroscopy was used to explore the nature of these clusters. We show that Myr-Src (2-9) is able to oligomerize, and the peptide clusters are embedded in a lipid platform generated by lipid sorting. Further, using confocal fluorescence microscopy and FRET assays we show that localized charge enrichment and membrane curvature are able to shift the partition coefficient towards the more ordered lipid phase.

Small-Molecule Inhibition of the UNC119-Cargo Interaction.

N-Terminal myristoylation facilitates membrane binding and activity of proteins, in particular of Src family kinases, but the underlying mechanisms are only beginning to be understood. The chaperones UNC119A/B regulate the cellular distribution and signaling of N-myristoylated proteins. Selective small-molecule modulators of the UNC119-cargo interaction would be invaluable tools, but have not been reported yet. We herein report the development of the first UNC119-cargo interaction inhibitor, squarunkin A. Squarunkin A selectively inhibits the binding of a myristoylated peptide representing the N-terminus of Src kinase to UNC119A with an IC50 value of 10 nm. It binds to UNC119 proteins in cell lysate and interferes with the activation of Src kinase. Our results demonstrate that small-molecule inhibition of the UNC119-cargo interaction might provide new opportunities for modulating the activity of Src kinases that are independent of direct inhibition of the enzymatic kinase activity.

Novel Biochemical and Structural Insights into the Interaction of Myristoylated Cargo with Unc119 Protein and Their Release by Arl2/3.

Primary cilia are highly specialized small antenna-like cellular protrusions that extend from the cell surface of many eukaryotic cell types. The protein content inside cilia and cytoplasm is very different, but details of the sorting process are not understood for most ciliary proteins. Recently, we have shown that prenylated proteins are sorted according to their affinity to the carrier protein PDE6δ and the ability of Arl3 but not Arl2 to release high affinity cargo inside the cilia (Fansa, E. K., Kösling, S. K., Zent, E., Wittinghofer, A., and Ismail, S. (2016) Nat. Commun. 7, 11366). Here we address the question whether a similar principle governs the transport of myristoylated cargo by the carrier proteins Unc119a and Unc119b. We thus analyzed the binding strength of N-terminal myristoylated cargo peptides (GNAT1, NPHP3, Cystin1, RP2, and Src) to Unc119a and Unc119b proteins. The affinity between myristoylated cargo and carrier protein, Unc119, varies between subnanomolar and micromolar. Peptides derived from ciliary localizing proteins (GNAT1, NPHP3, and Cystin1) bind with high affinity to Unc119 proteins, whereas a peptide derived from a non-ciliary localizing protein (Src) has low affinity. The peptide with intermediate affinity (RP2) is localized at the ciliary transition zone as a gate keeper. We show that the low affinity peptides are released by both Arl2·GppNHp and Arl3·GppNHp, whereas the high affinity peptides are exclusively released by only Arl3·GppNHp. Determination of the x-ray structure of myristoylated NPHP3 peptide in complex with Unc119a reveals the molecular details of high affinity binding and suggests the importance of the residues at the +2 and +3 positions relative to the myristoylated glycine for high and low affinities. The mutational analysis of swapping the residues at the +2 and +3 positions between high and low affinity peptides results in reversing their affinities for Unc119a and leads to a partial mislocalization of a low affinity mutant of NPHP3.

Synthesis of Lipidated Proteins.

Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented.

Specificity of Lipoprotein Chaperones for the Characteristic Lipidated Structural Motifs of their Cognate Lipoproteins.

Lipoprotein-binding chaperones mediate intracellular transport of lipidated proteins and determine their proper localisation and functioning. Understanding of the exact structural parameters that determine recognition and transport by different chaperones is of major interest. We have synthesised several lipid-modified peptides, representative of different lipoprotein classes, and have investigated their binding to the relevant chaperones PDEδ, UNC119a, UNC119b, and galectins-1 and -3. Our results demonstrate that PDEδ recognises S-isoprenylated C-terminal peptidic structures but not N-myristoylated peptides. In contrast, UNC119 proteins bind only mono-N-myristoylated, but do not recognise doubly lipidated and S-isoprenylated peptides at the C terminus. For galectins-1 and -3, neither binding to N-acylated, nor to C-terminally prenylated peptides could be determined. These results shed light on the specificity of the chaperone-mediated cellular lipoprotein transport systems.

All-carbon quaternary stereogenic centers in acyclic systems through the creation of several C-C bonds per chemical step.

In the past few decades, it has become clear that asymmetric catalysis is one of the most powerful methods for the construction of carbon-carbon as well as carbon-heteroatom bonds in a stereoselective manner. However, when structural complexity increases (i.e., all-carbon quaternary stereogenic center), the difficulty in reaching the desired adducts through asymmetric catalytic reactions leads to a single carbon-carbon bond-forming event per chemical step between two components. Issues of efficiency and convergence should therefore be addressed to avoid extraneous chemical steps. In this Perspective, we present approaches that tackle the stimulating problem of efficiency while answering interesting synthetic challenges. Ideally, if one could create all-carbon quaternary stereogenic centers via the creation of several new carbon-carbon bonds in an acyclic system and in a single-pot operation from simple precursors, it would certainly open new horizons toward solving the synthetic problems. Even more important for any further design, the presence of polyreactive intermediates in synthesis (bismetalated, carbenoid, and oxenoids species) becomes now an indispensable tool, as it creates consecutively the same number of carbon-carbon bonds as in a multi-step process, but in a single-pot operation.

Axial preferences in allylation reactions via the Zimmerman-Traxler transition state.

The reaction of a substituted allylmetal with a prostereogenic carbonyl compound can give rise to up to two racemic diastereomers (syn and anti). Classically, in such reactions, when pure E-isomers have afforded anti-selectivity and the Z-isomers exhibit syn-selectivity, researchers have used the empirical Zimmerman-Traxler model. In this model, chair-like transition states dominate over boat-like arrangements. The incoming aldehyde alkyl (aryl) residue occupies a pseudoequatorial rather than a pseudoaxial position to avoid potential 1,3-diaxial steric interactions. However, the reaction of γ,γ-disubstituted allylzinc species with carbonyl compounds generates two gauche interactions, which may result in a completely different stereochemical outcome. With these two gauche interactions, would a transition state in which the aldehyde substituent occupies a pseudoequatorial position or a pseudoaxial position be preferred? In this Account, we show that reaction of γ,γ-disubstituted allylzinc species with carbonyl compounds proceeds through a chair-like transition state and the substituent of the incoming aldehyde residue prefers to occupy a pseudoaxial position to avoid these two gauche interactions. Theoretical calculations on model systems support our experimental results. We have extended this new stereochemical outcome to describe the formation of α-alkoxyallylation of aldehydes through the formation of the rather uncommon (E)-γ,γ-disubstituted alkoxyallylzinc species. We also used this method to transform aromatic ketones and α-alkoxyaldehydes and ketones into functionalized adducts. In a one-pot reaction and using simple alkynes, three new carbon-carbon bonds and two to three stereogenic centers, including an all-carbon quaternary stereocenter could be created in acyclic systems. Because 1,3-diaxial interactions are now produced with the axial substituent, an increase in the substituent size on the zinc atom decreases the diastereoselectivity.

Convergent diastereoselective preparation of adjacent quaternary stereocenters in an acyclic system.

The combined carbometalation-zinc homologation-allylation reaction of the resulting stereodefined 3,3-disubstituted allylmetal species with ketones allow the preparation of allylic vicinal diol derivatives in good yields with excellent diastereomeric ratios from commercially available alkynes. Two adjacent quaternary centers are formed with the concomitant formation of three new carbon-carbon bonds in a single-pot operation in an acyclic system. The bulky substituent of the ketone occupies a pseudo-axial position in the Zimmerman-Traxler transition state.

Potential for phosphite and phosphonate utilization by Prochlorococcus.

Phosphonates (Pn) are diverse organic phosphorus (P) compounds containing C-P bonds and comprise up to 25% of the high-molecular weight dissolved organic P pool in the open ocean. Pn bioavailability was suggested to influence markedly bacterial primary production in low-P areas. Using metagenomic data from the Global Ocean Sampling expedition, we show that the main potential microbial contributor in Pn utilization in oceanic surface water is the globally important marine primary producer Prochlorococcus. Moreover, a number of Prochlorococcus strains contain two distinct putative Pn uptake operons coding for ABC-type Pn transporters. On the basis of microcalorimetric measurements, we find that each of the two different putative Pn-binding protein (PhnD) homologs transcribed from these operons possesses different Pn- as well as inorganic phosphite-binding specificities. Our results suggest that Prochlorococcus adapt to low-P environments by increasing the number of Pn transporters with different specificities towards phosphite and different Pns.

Combined carbometalation-zinc homologation-allylation reactions as a new approach for alkoxyallylation of aldehydes.

The combined carbometalation reaction of ynol ethers followed by a zinc homologation and further allylation reactions lead to an efficient preparation of allylic vicinal diols. The stereochemical outcome of the reaction shows that the substituent of the aldehyde occupies a pseudoaxial position in a Zimmerman-Traxler transition state.

Diverse functionalization of corannulene: easy access to pentagonal superstructure.

A variety of functionalized penta-arylcorannulene derivatives were prepared in high yields and high chemoselectivity by a cross-coupling reaction between sym-pentachlorocorannulene and substituted arylboronic acids using Fu's Pd(0) catalyst. This approach provides a general entry to penta-substituted corannulene derivatives, which are useful building blocks for various structures of high complexity, such as pentagonal dendrimers, synthetic capsids, and discotic liquid crystals. This was demonstrated here by the facile synthesis of a third generation pentagonal dendrimer.

Repulsive interaction can be a key design element of molecular rotary motors.

Low-barrier molecular rotary motors having rotaxane architecture can be constructed using a cucurbituril host and a polyyne guest serving as stator and rotator, respectively. The repulsive interaction between these components is supported by molecular mechanics calculations with model systems and experimentally verified by X-ray crystallography with several synthetic host-guest complexes, all suggesting that the diyne rod floats at the center of the macrocyclic host with no apparent van der Waals contacts between them. Further support for these interactions is suggested by microcalorimetry measurements.