Regulating molecule proximity: The physical distance, or proximity, between molecules often directs biological events. The development of membrane-permeable small molecules that reversibly regulate proximity has enabled advances in fields such as synthetic biology, signal transduction, transcription, protein degradation, epigenetic memory, and chromatin dynamics. This “induced proximity” can also be applied to the development of new therapeutics. Stanton et al. review the wide range of advances and speculate on future applications of this fundamental approach. Science , this issue p. eaao5902

Small-molecule inhibition of extracellular proteins that activate membrane receptors has proven to be extremely challenging. Diversity-oriented synthesis and small-molecule microarrays enabled the discovery of robotnikinin, a small molecule that binds the extracellular Sonic hedgehog (Shh) protein and blocks Shh signaling in cell lines, human primary keratinocytes and a synthetic model of human skin. Shh pathway activity is rescued by small-molecule agonists of Smoothened, which functions immediately downstream of the Shh receptor Patched.

Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi–histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD⁺) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG.

Sonic hedgehog (Shh) is the most widely characterized of the three vertebrate Hedgehoghomologs, and is essential for proper embryonic development. Shh binds to its receptor, Patched (Ptch1), resulting in the de-repression of Smoothened (Smo). This leads to the activation of Gli2, which regulates the transcription of target genes that include Gli1 and Ptch1. Several synthetic and naturally occurring small-molecule modulators of Smo have been discovered. Shh-signalingantagonists that bind to Smo include cyclopamine, SANT1, and Cur-61414. Shh signalingagonists that bind to Smo include the synthetic small molecules purmorphamine and SAG. Small molecules that inhibit Shh signalingdownstream of Smo, GANT58 and GANT61 have also been reported. Robotnikinin inhibits the Shh pathway by directly targeting Shh. Although progress has been made in understanding and modulating Shh signaling, fundamental aspects of Shh signal transduction remain obscure, including the mechanism(s) whereby Ptch1 regulates Smo activity. Small-molecule modulators of Shh signaling provide a means to regulate the activity of a pathway implicated in medulloblastoma, basal cell carcinoma (BCC), pancreatic cancer, prostate cancer and developmental disorders. Several Shh inhibitors have not succeeded in the clinic for unknown reasons, but clinical trials in BCC and pancreatic cancer with the promising Smo antagonists GDC-0449 and IPI-926 are currently underway.