Determination of RNA-ligand interactions with the photoaffinity platform PEARL-seq.

In the development of therapeutics, it is important to establish engagement of a compound to its intended target and identify other targets it binds to. Methods for demonstrating target engagement in the growing field of RNA-targeted therapeutics are therefore needed. We present a detailed protocol for Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), a platform for determining interactions between small molecule ligands and their target RNA(s). PEARL-seq allows detection of binding and crosslinking events with single nucleotide resolution and allows measurement of enrichment of the target RNA relative to all other RNAs. PEARL-seq is a valuable tool in the effort to verify bona fide RNA-ligand interactions.

Discovery and optimization of covalent Bcl-xL antagonists.

Over the last ten years, targeted covalent inhibition has become a key discipline within medicinal chemistry research, most notably in the development of oncology therapeutics. One area where this approach is underrepresented, however, is in targeting protein-protein interactions. This is primarily because these hydrophobic interfaces lack appropriately located cysteine residues to allow for standard conjugate addition chemistry. Herein, we report our development of the first covalent inhibitors of the antiapoptotic protein B-cell lymphoma extra-large (Bcl-xL), utilizing a sulfonyl fluoride (SF) warhead to selectively covalently modify tyrosine 101 of the BH3 domain-binding groove. These compounds display time-dependent inhibition in a biochemical assay and are cellularly active (U266B1). In addition, compound 7 was further elaborated to generate a chemical-biology probe molecule, which may find utility in understanding the intricacies of Bcl-xL biology.

Practical asymmetric conjugate alkynylation of Meldrum's acid-derived acceptors: access to chiral beta-alkynyl acids.

The enantioselective conjugate addition of alkynyl nucleophiles has been a long-standing challenge in synthetic chemistry. This paper describes a highly practical asymmetric conjugate alkynylation of Meldrum's acid-derived acceptors using cinchonidine (<$100/kg) as the chiral mediator. The process provides practical access to chiral beta-alkynyl acids. Noteworthy attributes of the method are its broad scope, high functional-group compatibility, and ease of scalability.

Rapid synthesis of an electron-deficient t-BuPHOX ligand: cross-coupling of aryl bromides with secondary phosphine oxides.

Herein an efficient and direct copper-catalyzed coupling of oxazoline-containing aryl bromides with electron-deficient secondary phosphine oxides is reported. The resulting tertiary phosphine oxides can be reduced to prepare a range of PHOX ligands. The presented strategy is a useful alternative to known methods for constructing PHOX derivatives.

PEARL-seq: A Photoaffinity Platform for the Analysis of Small Molecule-RNA Interactions.

RNA is emerging as a valuable target for the development of novel therapeutic agents. The rational design of RNA-targeting small molecules, however, has been hampered by the relative lack of methods for the analysis of small molecule-RNA interactions. Here, we present our efforts to develop such a platform using photoaffinity labeling. This technique, termed Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), enables the rapid identification of small molecule binding locations within their RNA targets and can provide information on ligand selectivity across multiple different RNAs. These data, when supplemented with small molecule SAR data and RNA probing data enable the construction of a computational model of the RNA-ligand structure, thereby enabling the rational design of novel RNA-targeted ligands.

Beyond cysteine: recent developments in the area of targeted covalent inhibition.

Over the past decade targeted covalent inhibitors have undergone a renaissance due to the clinical validation and regulatory approval of several small molecule therapeutics that are designed to irreversibly modify their target protein. Invariably, these compounds rely on the serendipitous placement of a cysteine residue proximal to the small molecule binding site; while this strategy has afforded numerous successes, it necessarily limits the number of proteins that can be targeted by this approach. This drawback has led several research groups to develop novel methodologies that target non-cysteine residues for covalent modification. Herein, we survey the current literature of warheads that covalently modify non-cysteine amino acids in proteins.

Anti-proliferative activity of the NPM1 interacting natural product avrainvillamide in acute myeloid leukemia.

Mutated nucleophosmin 1 (NPM1) acts as a proto-oncogene and is present in ~30% of patients with acute myeloid leukemia (AML). Here we examined the in vitro and in vivo anti-leukemic activity of the NPM1 and chromosome region maintenance 1 homolog (CRM1) interacting natural product avrainvillamide (AVA) and a fully syntetic AVA analog. The NPM1-mutated cell line OCI-AML3 and normal karyotype primary AML cells with NPM1 mutations were significantly more sensitive towards AVA than cells expressing wild-type (wt) NPM1. Furthermore, the presence of wt p53 sensitized cells toward AVA. Cells exhibiting fms-like tyrosine kinase 3 (FLT3) internal tandem duplication mutations also displayed a trend toward increased sensitivity to AVA. AVA treatment induced nuclear retention of the NPM1 mutant protein (NPMc+) in OCI-AML3 cells and primary AML cells, caused proteasomal degradation of NPMc+ and the nuclear export factor CRM1 and downregulated wt FLT3 protein. In addition, both AVA and its analog induced differentiation of OCI-AML3 cells together with an increased phagocytotic activity and oxidative burst potential. Finally, the AVA analog displayed anti-proliferative activity against subcutaneous xenografted HCT-116 and OCI-AML3 cells in mice. Our results demonstrate that AVA displays enhanced potency against defined subsets of AML cells, suggesting that therapeutic intervention employing AVA or related compounds may be feasible.

Interactions of the natural product (+)-avrainvillamide with nucleophosmin and exportin-1 Mediate the cellular localization of nucleophosmin and its AML-associated mutants.

Nucleophosmin (NPM1) is a multifunctional phosphoprotein localized predominantly within the nucleoli of eukaryotic cells. Mutations within its C-terminal domain are frequently observed in patients with acute myeloid leukemia (AML), are thought to play a key role in the initiation of the disease, and result in aberrant, cytoplasmic localization of the mutant protein. We have previously shown that the electrophilic antiproliferative natural product (+)-avrainvillamide (1) binds to proteins, including nucleophosmin, by S-alkylation of cysteine residues. Here, we report that avrainvillamide restores nucleolar localization of certain AML-associated mutant forms of NPM1 and provide evidence that this relocalization is mediated by interactions of avrainvillamide with mutant NPM1 and exportin-1 (Crm1). Immunofluorescence and mass spectrometric experiments employing a series of different NPM1 constructs suggest that a specific interaction between avrainvillamide and Cys275 of certain NPM1 mutants mediates the relocalization of these proteins to the nucleolus. Avrainvillamide treatment is also shown to inhibit nuclear export of Crm1 cargo proteins, including AML-associated NPM1 mutants. We also observe that avrainvillamide treatment displaces Thr199-phosphorylated NPM1 from duplicated centrosomes, leads to an accumulation of supernumerary centrosomes, and inhibits dephosphorylation of Thr199-phosphorylated NPM1 by protein phosphatase 1. Avrainvillamide is the first small molecule reported to relocalize specific cytoplasmic AML-associated NPM1 mutants to the nucleolus, providing an important demonstration of principle that small molecule induction of a wild-type NPM1 localization phenotype is feasible in certain human cancer cells.

A catalytic, asymmetric formal synthesis of (+)-hamigeran B.

A concise asymmetric, formal synthesis of (+)-hamigeran B is reported. A Pd-catalyzed, decarboxylative allylic alkylation, employing a trifluoromethylated derivative of t-BuPHOX, is utilized as the enantioselective step to form the critical quaternary carbon center in excellent yield and enantioselectivity. The product is converted in three steps to a late-stage intermediate previously used in the synthesis of hamigeran B.