Lisa A. Marcaurelle

Identifying the proteins to which small-molecule probes and drugs bind in cells

Most small-molecule probes and drugs alter cell circuitry by interacting with 1 or more proteins. A complete understanding of the interacting proteins and their associated protein complexes, whether the compounds are discovered by cell-based phenotypic or target-based screens, is extremely rare. Such a capability is expected to be highly illuminating—providing strong clues to the mechanisms used by small-molecules to achieve their recognized actions and suggesting potential unrecognized actions. We describe a powerful method combining quantitative proteomics (SILAC) with affinity enrichment to provide unbiased, robust and comprehensive identification of the proteins that bind to small-molecule probes and drugs. The method is scalable and general, requiring little optimization across different compound classes, and has already had a transformative effect on our studies of small-molecule probes. Here, we describe in full detail the application of the method to identify targets of kinase inhibitors and immunophilin binders.

An aldol-based build/couple/pair strategy for the synthesis of medium- and large-sized rings: discovery of macrocyclic histone deacetylase inhibitors.

An aldol-based build/couple/pair (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the build phase, a series of asymmetric syn- and anti-aldol reactions were performed to produce four stereoisomers of a Boc-protected γ-amino acid. In addition, both stereoisomers of O-PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the couple step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the pair phase, three different reactions were employed to enable intramolecular ring-forming processes: nucleophilic aromatic substitution (S(N)Ar), Huisgen [3+2] cycloaddition, and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields, providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on the solid phase to yield a 14 400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition, and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.

Grand Challenge Commentary: Accessing new chemical space for 'undruggable' targets

The synthesis and biological annotation of small molecules from underexplored chemical space will play a central role in the development of drugs for challenging targets currently being identified in frontier areas of biological research such as human genetics.

New Directions in the Synthesis of Glycopeptide Mimetics

Structural and functional mimetics of naturally occurring glycopeptides are attractive synthetic targets owing to their potential as biological tools and therapeutic agents. The syntheses of C- and S-glycopeptides, glycopeptoids, and oxime- (figure) and thioether-linked glycopeptides have now been realized, and many of these mimetics are currently undergoing biological evaluation.

Chemical Synthesis of Lymphotactin: A Glycosylated Chemokine with a C-Terminal Mucin-Like Domain

The synthesis of a 93-residue chemokine, lymphotactin, containing eight sites of O-linked glycosylation, was achieved using the technique of native chemical ligation. A single GalNAc residue was incorporated at each glycosylation site using standard Fmoc-chemistry to achieve the first total synthesis of a mucin-type glycoprotein. Using this approach quantities of homogeneous material were obtained for structural and functional analysis.

Accessing Skeletal Diversity Using Catalyst Control: Formation of n and n + 1 Macrocyclic Triazole Rings

A regioselective intramolecular Huisgen cycloaddition was performed on various azido alkyne substrates giving rise to macrocyclic triazole rings. Using catalyst control, a common intermediate has been converted to two structurally unique macrocycles with either a 1,5- or a 1,4-triazole resulting in an n or n + 1 ring size. This is the first example of an intramolecular ruthenium-catalyzed Huisgen cycloaddition.

Synthesis and Profiling of a Diverse Collection of Azetidine-Based Scaffolds for the Development of CNS-Focused Lead-like Libraries

The synthesis and diversification of a densely functionalized azetidine ring system to gain access to a wide variety of fused, bridged, and spirocyclic ring systems is described. The in vitro physicochemical and pharmacokinetic properties of representative library members are measured in order to evaluate the use of these scaffolds for the generation of lead-like molecules to be used in targeting the central nervous system. The solid-phase synthesis of a 1976-membered library of spirocyclic azetidines is also described.

Combinatorial carbohydrate chemistry

The application of combinatorial chemistry to the synthesis of carbohydrate-based compound collections has received increased attention in recent years. New strategies for the solution-phase synthesis of oligosaccharide libraries have been reported, and the use of monosaccharides as scaffolds in the generation of combinatorial libraries has been described. Novel approaches to the assembly of carbohydrate-based antibiotics, such as aminoglycoside analogs and vancomycin derivatives, have also been disclosed.

Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure–activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.

Synthesis of an oxime-linked neoglycopeptide with glycosylation-dependent activity similar to its native counterpart

Abstract Neoglycopeptides containing an oxime sugar-peptide linkage can be generated by coupling an aminooxy sugar with a peptide bearing a keto-amino acid. The coupling reaction can be executed in aqueous milieu without need for protecting groups on the peptide or saccharide, or auxiliary coupling reagents. Using this method, an oxime-linked analog of an antimicrobial peptide with glycosylation-dependent function was prepared and found to have similar bioactivity to the native glycopeptide. Thus, replacement of the sugar-peptide bond with an unnatural but synthetically facile linkage can produce functional neoglycopeptides.