Discovery of Sulanemadlin (ALRN-6924), the First Cell-Permeating, Stabilized α-Helical Peptide in Clinical Development.

We report the discovery of sulanemadlin (ALRN-6924), the first cell-permeating, stabilized α-helical peptide to enter clinical trials. ALRN-6924 is a "stapled peptide" that mimics the N-terminal domain of the p53 tumor suppressor protein. It binds with high affinity to both MDM2 and MDMX (also known as MDM4), the endogenous inhibitors of p53, to activate p53 signaling in cells having a non-mutant, or wild-type TP53 genotype (TP53-WT). Iterative structure-activity optimization endowed ALRN-6924 with favorable cell permeability, solubility, and pharmacokinetic and safety profiles. Intracellular proteolysis of ALRN-6924 forms a long-acting active metabolite with potent MDM2 and MDMX binding affinity and slow dissociation kinetics. At high doses, ALRN-6924 exhibits on-mechanism anticancer activity in TP53-WT tumor models. At lower doses, ALRN-6924 transiently arrests the cell cycle in healthy tissues to protect them from chemotherapy without protecting the TP53-mutant cancer cells. These results support the continued clinical evaluation of ALRN-6924 as an anticancer and chemoprotection agent.

Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression.

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases.

Stapled α-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy.

Stapled α-helical peptides have emerged as a promising new modality for a wide range of therapeutic targets. Here, we report a potent and selective dual inhibitor of MDM2 and MDMX, ATSP-7041, which effectively activates the p53 pathway in tumors in vitro and in vivo. Specifically, ATSP-7041 binds both MDM2 and MDMX with nanomolar affinities, shows submicromolar cellular activities in cancer cell lines in the presence of serum, and demonstrates highly specific, on-target mechanism of action. A high resolution (1.7-Å) X-ray crystal structure reveals its molecular interactions with the target protein MDMX, including multiple contacts with key amino acids as well as a role for the hydrocarbon staple itself in target engagement. Most importantly, ATSP-7041 demonstrates robust p53-dependent tumor growth suppression in MDM2/MDMX-overexpressing xenograft cancer models, with a high correlation to on-target pharmacodynamic activity, and possesses favorable pharmacokinetic and tissue distribution properties. Overall, ATSP-7041 demonstrates in vitro and in vivo proof-of-concept that stapled peptides can be developed as therapeutically relevant inhibitors of protein-protein interaction and may offer a viable modality for cancer therapy.

STAT6 phosphorylation inhibitors block eotaxin-3 secretion in bronchial epithelial cells.

The STAT6 (signal transducer and activator of transcription 6) protein facilitates T-helper cell 2 (Th2) mediated responses that control IgE-mediated atopic diseases such as asthma. We have identified compounds that bind to STAT6 and inhibit STAT6 tyrosine phosphorylation induced by IL-4. In the bronchial epithelial cell line BEAS-2B, compound (R)-84 inhibits the secretion of eotaxin-3, a chemokine eliciting eosinophil infiltration. (R)-84 appears to prevent STAT6 from assuming the active dimer configuration by directly binding the protein and inhibiting tyrosine phosphorylation.

Inhibitors of the lipid phosphatase SHIP2 discovered by high-throughput affinity selection-mass spectrometry screening of combinatorial libraries.

This manuscript describes the discovery and characterization of inhibitors of the lipid phosphatase SHIP2, an important target for the treatment of Type 2 diabetes, using the Automated Ligand Identification System. ALIS is an affinity selection-mass spectrometry platform for label-free, high throughput screening of mixture-based combinatorial libraries. We detail the mass-encoded synthesis of a library that yielded NGD-61338, a pyrazole-based SHIP2 inhibitor. Quantitative ALIS affinity measurements and inhibition of SHIP2 enzymatic activity indicate that this compound has micromolar binding affinity and inhibitory activity for this target. This inhibitor, which does not contain a phosphatase "warhead," binds the active site of SHIP2 as determined by ALIS-based competition experiments with the enzyme's natural substrate, phosphatidylinositol 3,4,5-triphosphate (PIP3). Structure-activity relationships for NGD-61338 and two other ligand classes discovered by ALIS screening were explored using a combination of combinatorial library synthesis and ALIS-enabled affinity ranking in compound mixtures.

Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry.

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.

A general technique to rank protein-ligand binding affinities and determine allosteric versus direct binding site competition in compound mixtures.

To realize the full potential of combinatorial chemistry-based drug discovery, generic and efficient tools must be developed that apply the strengths of diversity-oriented chemical synthesis to the identification and optimization of lead compounds for disease-associated protein targets. We report an affinity selection-mass spectrometry (AS-MS) method for protein-ligand affinity ranking and the classification of ligands by binding site. The method incorporates the following steps: (1) an affinity selection stage, where protein-binding compounds are selected from pools of ligands in the presence of varying concentrations of a competitor ligand, (2) a first chromatography stage to separate unbound ligands from protein-ligand complexes, and (3) a second chromatography stage to dissociate the ligands from the complexes for identification and quantification by MS. The ability of the competitor ligand to displace a target-bound library member, as measured by MS, reveals the binding site classification and affinity ranking of the mixture components. The technique requires no radiolabel incorporation or direct biochemical assay, no modification or immobilization of the compounds or target protein, and all reaction components, including any buffers or cofactors required for protein stability, are free in solution. We demonstrate the method for several compounds of wide structural variety against representatives of the most important protein classes in contemporary drug discovery, including novel ATP-competitive and allosteric inhibitors of the Akt-1 (PKB) and Zap-70 kinases, and previously undisclosed antagonists of the M(2) muscarinic acetylcholine receptor, a G-protein coupled receptor (GPCR). The theoretical basis of the technique is analyzed mathematically, allowing quantitative estimation of binding affinities and, in the case of allosteric interaction, absolute determination of binding cooperativity. The method is readily applicable to high-throughput screening hit triage, combinatorial library-based affinity optimization, and developing structure-activity relationships among multiple ligands to a given receptor.

Identification of a small molecule nonpeptide active site beta-secretase inhibitor that displays a nontraditional binding mode for aspartyl proteases.

A small molecule nonpeptide inhibitor of beta-secretase has been developed, and its binding has been defined through crystallographic determination of the enzyme-inhibitor complex. The molecule is shown to bind to the catalytic aspartate residues in an unprecedented manner in the field of aspartyl protease inhibition. Additionally, the complex reveals a heretofore unknown S(3) subpocket that is created by the inhibitor. This structure has served an important role in the design of newer beta-secretase inhibitors.

A reagent-based strategy for the design of large combinatorial libraries: a preliminary experimental validation.

Combinatorial library design can be carried out at either the reagent or the product level. Various reports in the literature have come to conflicting conclusions in favor of one over the other. In this paper a reagent-based screening library design strategy is presented. The method relies on analysis of scaffolds and building blocks separately to define the overall diversity in a compound file. The primary diversity selection by properties relevant for molecular recognition and by redundancy is followed by the application of filters for molecular properties known to be relevant for drug-likeness. Filter properties are rapidly estimated at the product level using a fragmental estimation approach. Initial experimental data suggest that high diversity in vast screening libraries can be achieved by carefully applied reagent level analysis. A potential role of diverse screening libraries in chemical genomics (pharmacological knockouts) is also discussed.