Total synthesis of cruentaren A

Cruentaren A, a highly cytotoxic metabolite, which also inhibits F-ATPase, was synthesized using our recently developed methodology on resorcylic acid lactones natural products. [Molecular structure diagrams appear here. To view, please open pdf attachment] Alcohol A was prepared on a multigram scale in 13 steps starting from (S)-Roche ester and using highly stereoselective reactions such as Evans aldol reaction and asymmetric propargylation. [Molecular structure diagrams appear here. To view, please open pdf attachment] Key fragment B was synthesized in 11 steps from 1,3-propanediol. The 1,2-anti-configuration was installed with a Brown crotylation. Diketo-dioxinone D was generated from C-acylation between Weinreb amide B and keto-dioxinone C. Ketene generation by thermolysis followed by trapping with alcohol A and aromatization afforded resorcylate derivative E. [Molecular structure diagrams appear here. To view, please open pdf attachment] Finally after a sequence consisting of the following key steps: ring closing alkyne metathesis, coupling between amine G and acid H and Lindlar hydrogenation, cruentaren A was obtained. [Molecular structure diagrams appear here. To view, please open pdf attachment

Estimating intrinsic cooperativities and concentrations of ternary complexes in biochemical or cellular environments from binary dissociation constants, apparent cooperativities and total or free ligand concentrations

There is an increasing interest to modulate difficult-to-tackle but disease-relevant target proteins by involving them with a chaperone protein into a compound-mediated ternary complex. In general, ternary complex-forming compounds can be classified by their direct affinity to chaperone and target protein and their degree of cooperativity which they exhibit upon ternary complex formation. As a trend, the smaller ternary complex-forming compounds are the stronger is the contribution of the intrinsic cooperativity to their thermodynamic stability relative to direct target (or chaperone) binding. This highlights the importance of the cooperativity as a desirable feature for the optimization of ternary complex-forming compounds – even more so as it provides higher and easier to achieve selectivity to target isoforms and allows the assessment of target occupancy via estimated concentrations of ternary complexes. All of this emphasizes the need to quantify the natural constant of intrinsic cooperativity α which is defined as the gain (or loss) in affinity of a compound to its target in pre-bound vs. unbound state. In this publication, a workflow involving a mathematical model is presented that requires the input of the two relevant binary Kds and the two protein concentrations of target and chaperone for estimating the intrinsic cooperativity from experimentally observed apparent cooperativities. Intrinsic cooperativities are in the simplest version assessable through EC50 shifts in binary binding curve(s) of the ternary complex-forming compound with either target or chaperone relative to the same experiment but in the presence of also the counter protein. This approach is then extended from closed systems (like biochemical assays) to the open system of a cellular assay by accounting for differences in total ligand vs. free ligand concentrations, which then allows calculating ternary complexes concentrations. Finally, this model is used to translate biochemical potency of ternary complex-forming compounds into expected cellular target occupancy, which could ultimately serve as way for validation or de-validation of hypothesized biological mechanisms of action

A model-informed method to retrieve intrinsic from apparent cooperativity and project cellular target occupancy for ternary complex-forming compounds.

There is an increasing interest to develop therapeutics that modulate challenging or undruggable target proteins via a mechanism that involves ternary complexes. In general, such compounds can be characterized by their direct affinities to a chaperone and a target protein and by their degree of cooperativity in the formation of the ternary complex. As a trend, smaller compounds have a greater dependency on intrinsic cooperativity to their thermodynamic stability relative to direct target (or chaperone) binding. This highlights the need to consider intrinsic cooperativity of ternary complex-forming compounds early in lead optimization, especially as they provide more control over target selectivity (especially for isoforms) and more insight into the relationship between target occupancy and target response via estimation of ternary complex concentrations. This motivates the need to quantify the natural constant of intrinsic cooperativity (α) which is generally defined as the gain (or loss) in affinity of a compound to its target in pre-bound vs. unbound state. Intrinsic cooperativities can be retrieved via a mathematical binding model from EC50 shifts of binary binding curves of the ternary complex-forming compound with either a target or chaperone relative to the same experiment but in the presence of the counter protein. In this manuscript, we present a mathematical modeling methodology that estimates the intrinsic cooperativity value from experimentally observed apparent cooperativities. This method requires only the two binary binding affinities and the protein concentrations of target and chaperone and is therefore suitable for use in early discovery therapeutic programs. This approach is then extended from biochemical assays to cellular assays (i.e., from a closed system to an open system) by accounting for differences in total ligand vs. free ligand concentrations in the calculations of ternary complex concentrations. Finally, this model is used to translate biochemical potency of ternary complex-forming compounds into expected cellular target occupancy, which could ultimately serve as a way for validation or de-validation of hypothesized biological mechanisms of action

Hydroxymethyl salicylaldehyde-promoted ligation: a new functionalization for chemoselective ligations

A new amide-forming ligation exhibiting an unprecedented selectivity for glycine is described herein. The distinguishing feature of this ligation is it's reliance on an ortho-hydroxymethyl salicylaldehyde ester at the C-terminus which allows, via an N,O-acetal intermediate, the formation of a native peptide bond

Polar/Apolar Interfaces Modulate the Conformational Behavior of Cyclic Peptides with Impact on Their Passive Membrane Permeability

Cyclic peptides have the potential to vastly extend the scope of druggable proteins and lead to new therapeutics for currently untreatable diseases. However, cyclic pep- tides often su�er from poor bioavailability. To uncover design principles for permeable cyclic peptides, a promising strategy is to analyze the conformational dynamics of the peptides using molecular dynamics (MD) and Markov state models (MSMs). Previous MD studies have focused on the conformational dynamics in pure aqueous or apolar environments to rationalize membrane permeability. However, during the key steps of the permeation through the membrane, cyclic peptides are exposed to interfaces be- tween polar and apolar regions. Recent studies revealed that these interfaces constitute the free energy minima of the permeation process. Thus, a deeper understanding of the behavior of cyclic peptides at polar/apolar interfaces is desired. Here, we investigate the conformational and kinetic behavior of cyclic decapeptides at a water/chloroform interface using unbiased MD simulations and MSMs. The distinct environments at the interface alter the conformational equilibrium as well as the interconversion kinetics of cyclic peptide conformations. For peptides with low population of the permeable con- formation in aqueous solution, the polar/apolar interface facilitates the interconversion to the closed conformation, which is required for membrane permeation. Comparison to unbiased MD simulations with a POPC bilayer reveals that not only the conforma- tions but also the orientations are relevant in a membrane system. These �ndings allow us to propose a permeability model that includes both 'prefolding' and 'non-prefolding' cyclic peptides - an extension that can lead to new design considerations for permeable cyclic peptides

Lessons for Oral Bioavailability: How Conformationally Flexible Cyclic Peptides Enter and Cross Lipid Membranes

Cyclic peptides are able to extend the druggable space of pharmaceutical targets, due to their size, conformational behavior, and high proportion of hydrogen bond donors and acceptors. However, for the same reasons, they often suffer from poor membrane permeation and thus low oral bioavailability. As permeability assays do not allow to monitor the pathway and behavior of cyclic peptides on their “journey” trough lipid membranes, little is known about the underlying permeation process, which poses a major obstacle for their rational design. Here, we use molecular dynamics (MD) simulations as a computational microscope to uncover how large and conformationally flexible cyclic peptides enter and cross a lipid bilayer. In a first step, we performed unbiased MD simulations to elucidate the permeation pathway. Subsequently, this pathway knowledge was utilized to seed biased simulations to further enrich for permeation events. Based on our simulations, we show how specific side-chain residues can act as ’molecular anchors’, which establish the first contact between the peptides and the membrane, and consequently enable membrane insertion. Inside the membrane, the cyclic peptides are positioned directly between the polar headgroup and the apolar tail region, where they are subjected to a unique polar/apolar interface environment. In this environment, the cyclic peptides show a preference for one of two distinct orientations. We observe that only one of these orientations allows the formation of the permeable ’closed’ conformation, and only in this ’closed’ conformation the cyclic peptides can cross from the upper to the lower membrane leaflet, which again requires a unique anchoring and flipping mechanism. Our findings provide atomistic insights into the permeation process of flexible cyclic peptides and reveal unique design considerations for each step of the process

Design and Development of a Cyclic Decapeptide Scaffold with Suitable Properties for Bioavailability and Oral Exposure

Permeability and oral bioavailability of macrocyclic peptides still represents a difficult challenge in drug discovery. Despite their recognised potential as therapeutics, their use is still restricted to extra-cellular targets and i.v. administration. Indeed, macrocyclic peptides generally suffer from limited proteolytic stability, high clearance and poor membrane permeability leading to the absence of systemic exposure after oral administration. In order to overcome these limitations, we started to investigate the development of a general cyclic decapeptide scaffold that possesses ideal features for cell permeability and oral exposure. Based on a hairpin structure, the scaffold design aimed to reduce the overall polarity of the compound thereby limiting the energetic cost of NH desolvation and the entropy penalty during cell penetration. The results of this study demonstrate the importance of rigidity for the -turn design regarding clearance. In order to stabilize the scaffold in the desired β-hairpin conformation to favour permeability, the introduction of D-proline at the i+1 turn position also proved to be beneficial in limiting clearance. As a result, cyclopeptide decamers with unprecedented high values for oral bioavailability and exposure are reported herein. NMR conformation and dynamic analysis confirmed for selected examples the rigidity of the scaffold and the presence of trans-annular hydrogen bonds in polar and apolar environments. Furthermore, we showed that the excellent bioavalability value obtained for one compound was supported by a favourable entropy for the transition from a polar to an apolar environment