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Fungi have historically been the source of numerous important medicinal compounds, but full exploitation of their genetic potential for drug development has been hampered in traditional discovery paradigms. Here we describe a radically different approach, top-down drug discovery (TD 3 ), starting with a massive digital search through a database of over 100,000 fully genomicized fungi to identify loci encoding molecules with a predetermined human target. We exemplify TD 3 by the selection of cyclin-dependent kinases (CDKs) as targets and the discovery of two molecules, 1 and 2 , which inhibit therapeutically important human CDKs. 1 and 2 exhibit a remarkable mechanism, forming a site-selective covalent bond to the CDK active site Lys. We explored the structure-activity relationship via semi- and total synthesis, generating an analog, 43 , with improved kinase selectivity, bioavailability, and efficacy. This work highlights the power of TD 3 to identify mechanistically and structurally novel molecules for the development of new medicines.

Huntington's disease (HD) is an autosomal dominant disease caused by the expansion of cytosine-adenine-guanine (CAG) repeats in one copy of the HTT gene (mutant HTT, mHTT). The unaffected HTT gene encodes wild-type HTT (wtHTT) protein, which supports processes important for the health and function of the central nervous system. Selective lowering of mHTT for the treatment of HD may provide a benefit over nonselective HTT-lowering approaches, as it aims to preserve the beneficial activities of wtHTT. Targeting a heterozygous single-nucleotide polymorphism (SNP) where the targeted variant is on the mHTT gene is one strategy for achieving allele-selective activity. Herein, we investigated whether stereopure phosphorothioate (PS)- and phosphoryl guanidine (PN)-containing oligonucleotides can direct allele-selective mHTT lowering by targeting rs362273 (SNP3). We demonstrate that our SNP3-targeting molecules are potent, durable, and selective for mHTT in vitro and in vivo in mouse models. Through comparisons with a surrogate for the nonselective investigational compound tominersen, we also demonstrate that allele-selective molecules display equivalent potency toward mHTT with improved durability while sparing wtHTT. Our preclinical findings support the advancement of WVE-003, an investigational allele-selective compound currently in clinical testing (NCT05032196) for the treatment of patients with HD., Competing Interests: N.I., Y.L., M.F.-K., A.M., W.C.T., K.T., N.K., A.A., K.B., J.D.S., A.L., X.S.H., H.G.J., P.K., F.L., K.L., R.L., M., J.M., Q.P., E.P.-E., M.S., P.S.P., S.S., H.Y., A.Z., C.F., M.B., E.D., and C.V. were employees of Wave Life Sciences during the completion of this work. M. is currently an employee of Stoke Therapeutics. G.L.V. is on the board of directors and is a consultant and shareholder for Wave Life Sciences., (© 2024 The Author(s).)

Widespread adoption of mirror-image biological systems presents difficulties in accessing the requisite D-protein substrates. In particular, mirror-image phage display has the potential for high-throughput generation of biologically stable macrocyclic D-peptide binders with potentially unique recognition modes but is hindered by the individualized optimization required for D-protein chemical synthesis. We demonstrate a general mirror-image phage display pipeline that utilizes automated flow peptide synthesis to prepare D-proteins in a single run. With this approach, we prepare and characterize 12 D-proteins - almost one third of all reported D-proteins to date. With access to mirror-image protein targets, we describe the successful discovery of six macrocyclic D-peptide binders: three to the oncoprotein MDM2, and three to the E3 ubiquitin ligase CHIP. Reliable production of mirror-image proteins can unlock the full potential of D-peptide drug discovery and streamline the study of mirror-image biology more broadly., (© 2024. The Author(s).)

Molecules that induce novel interactions between proteins hold great promise for the study of biological systems and the development of therapeutics, but their discovery has been limited by the complexities of rationally designing interactions between three components, and because known binders to each protein are typically required to inform initial designs. Here, we report a general and rapid method for discovering α-helically constrained (Helicon) polypeptides that cooperatively induce the interaction between two target proteins without relying on previously known binders or an intrinsic affinity between the proteins. We show that Helicons are capable of binding every major class of E3 ubiquitin ligases, which are of great biological and therapeutic interest but remain largely intractable to targeting by small molecules. We then describe a phage-based screening method for discovering "trimerizer" Helicons, and apply it to reprogram E3s to cooperatively bind an enzyme (PPIA), a transcription factor (TEAD4), and a transcriptional coactivator (β-catenin)., (© 2023. The Author(s).)

The α-helix is one of the most common protein surface recognition motifs found in nature, and its unique amide-cloaking properties also enable α-helical polypeptide motifs to exist in membranes. Together, these properties have inspired the development of α-helically constrained (Helicon) therapeutics that can enter cells and bind targets that have been considered "undruggable", such as protein-protein interactions. To date, no general method for discovering α-helical binders to proteins has been reported, limiting Helicon drug discovery to only those proteins with previously characterized α-helix recognition sites, and restricting the starting chemical matter to those known α-helical binders. Here, we report a general and rapid screening method to empirically map the α-helix binding sites on a broad range of target proteins in parallel using large, unbiased Helicon phage display libraries and next-generation sequencing. We apply this method to screen six structurally diverse protein domains, only one of which had been previously reported to bind isolated α-helical peptides, discovering 20 families that collectively comprise several hundred individual Helicons. Analysis of 14 X-ray cocrystal structures reveals at least nine distinct α-helix recognition sites across these six proteins, and biochemical and biophysical studies show that these Helicons can block protein-protein interactions, inhibit enzymatic activity, induce conformational rearrangements, and cause protein dimerization. We anticipate that this method will prove broadly useful for the study of protein recognition and for the development of both biochemical tools and therapeutics for traditionally challenging protein targets.

Aberrant activation of the Wnt signaling pathway has been identified in numerous types of cancer. One common feature of oncogenic Wnt regulation involves an increase in the cellular levels of β-catenin due to interference with its constitutive ubiquitin-dependent degradation. Targeting β-catenin has therefore emerged as an appealing approach for the treatment of Wnt-dependent cancers. Here, we report a strategy that employs multifunctional stapled peptides to recruit an E3 ubiquitin ligase to β-catenin, thereby rescuing β-catenin degradation by hijacking the endogenous ubiquitin-proteasome pathway. Specifically, we designed, synthesized, and evaluated a panel of multifunctional stapled peptides that have a β-catenin binding moiety (StAx-35) covalently linked to a second stapled peptide moiety (SAH-p53-8), which is capable to interact with the E3 ubiquitin ligase MDM2. We found that in vitro these multifunctional peptides can recruit the MDM2 protein to β-catenin and induce poly-ubiquitination on β-catenin. In cellulo, treatment of the human colorectal cancer cell line SW480 with the multifunctional stapled peptides showed dose-dependent degradation of endogenous β-catenin levels. In addition, a luciferase reporter assay showed that the multifunctional stapled peptides can suppress β-catenin-mediated gene expression via the Wnt signaling pathway. Therefore, these multifunctional stapled peptides provide a unique research tool for examining the Wnt signaling pathway by targeted knockdown of β-catenin at the protein level, and may serve as leads for potential drug candidates in the treatment of Wnt-dependent cancers., (© 2021 European Peptide Society and John Wiley & Sons, Ltd.)