Bifunctional modalities for repurposing protein function

Nature takes advantage of induced proximity to perform various functions. Taking inspiration from nature, we can also trigger desired biological processes using bifunctional small molecules that artificially induce proximity. For example, have been designed the ubiquitin-dependent proteasomal degradation intracellular proteins. Now, recent classes compounds developed degrade extracellular targets, membrane proteins, damaged organelles, and RNA by recruiting alternative pathways. In addition inducing degradation, modalities change phosphorylation glycosylation states evoke a response. this review, highlight advances in these innovative build on rich history chemical inducers dimerization. We anticipate more molecules, which or remove posttranslational modifications, endow neo-functionalities will emerge. Cellular homeostasis, key hallmark living organisms, arises interactions between biomolecules—especially enzyme-substrate interactions—within outside cell. Conventionally, function particular enzyme has investigated controlled through use an inhibitor, such (e.g., ATP-competitive kinase inhibitors) provided conceptual pathway toward many FDA-approved drugs (Figure 1A). However, inhibition is only one for altering function, cellular changes cannot be accomplished alone. Recently, there resurgence another class operate target evoking number functions beyond (Stanton et al., 2018Stanton B.Z. Chory E.J. Crabtree G.R. Chemically biology medicine.Science. 2018; 359: eaao5902Crossref PubMed Scopus (122) Google Scholar; Gerry Schreiber, 2020Gerry C.J. Schreiber S.L. Unifying principles bifunctional, proximity-inducing molecules.Nat. Chem. Biol. 2020; 16: 369-378Crossref (50) 2021Schreiber The rise molecular glues.Cell. 2021; 184: 3-9Abstract Full Text PDF (61) Scholar). Like conventional inhibitors, allow investigation are propelling development cutting-edge therapeutic modalities. These classified as “dumbbells” “molecular glues” 1B), where dumbbells link two individually bind their while glues similar ternary complexes without obvious linker. Conventional inhibitors form binary with protein exhibit different features compared (Hughes Ciulli, 2017Hughes S.J. Ciulli A. Molecular recognition complexes: new dimension structure-guided design degraders.Essays Biochem. 2017; 61: 505-516Crossref (96) Lai Crews, 2017Lai A.C. Crews C.M. Induced degradation: emerging drug discovery paradigm.Nat. Rev. Drug Discov. 101-114Crossref (593) complexes, activators functional site, either orthosterically allosterically, modulate sites, including active allosteric sites 1B). While occupancy driven, often event driven. As result, stoichiometric, sub-stoichiometric catalytic. Furthermore, require strong binding affinities, whereas may low-to-moderate affinities targeted some rely cooperativity. Another noteworthy comparison that, globally affect used localize enzymatic activity given target. addition, saturation effect, at high concentrations, site occupied. contrast, hook concentrations molecule saturate partners into individual resulting loss efficacy higher dose (Rodbard 1978Rodbard D. Feldman Y. Jaffe M.L. Miles L.E. Kinetics two-site immunoradiometric ('sandwich') assays-II. Studies nature 'high-dose effect.Immunochemistry. 1978; 15: 77-82Crossref (85) Mathematical frameworks describe three-body equilibria support experimental theoretical findings (Douglass 2013Douglass Jr., E.F. Miller Sparer G. Shapiro H. Spiegel D.A. A comprehensive mathematical model equilibria.J. Am. Soc. 2013; 135: 6092-6099Crossref (172) Bifunctional increase effective molarity components protein) arrange them meaningful orientations This underlying principle employed cells produce output scenarios modus operandi natural products cyclosporin A) involve induction proteins (Schreiber, Leveraging principle, Spencer al. “synthesized signaling pathway” rationally developing synthetic signal transducers (Spencer 1993Spencer D.M. Wandless T.J. Controlling transduction ligands.Science. 1993; 262: 1019-1024Crossref (681) work laid foundation wide array activities ranging specific modifications (PTMs). large high-throughput genomic sequencing TCGA ICGC) (ICGC/TCGA Pan-Cancer Analysis Whole Genomes Consortium, 2020ICGC/TCGA Consortium Pan-cancer analysis whole genomes.Nature. 578: 82-93Crossref (769) Scholar) RNAi CRISPR screening (DepMap) (Tsherniak 2017Tsherniak Vazquez F. Montgomery P.G. Weir B.A. Kryukov Cowley G.S. Gill S. Harrington W.F. Pantel Krill-Burger J.M. al.Defining cancer dependency map.Cell. 170: 564-576 e516Abstract (750) efforts suggest genetic variants drive human diseases via PTMs (Iqbal 2020Iqbal Perez-Palma E. Jespersen J.B. May P. Hoksza Heyne H.O. Ahmed S.S. Rifat Z.T. Rahman M.S. Lage K. al.Comprehensive characterization amino acid positions structures reveals effect missense variants.Proc. Natl. Acad. Sci. U S 117: 28201-28211Crossref (18) Scholar), instances, haploinsufficiency gene dosage enzymes could drivers. Such alterations cause biochemical hyperactivation receptor tyrosine kinases (RTKs; e.g., PI3KCA, MET, EGFR), frequently mutated cancer. ability caused mutation drivers provide options treating serious diseases. Here, review native neo-functions, beginning those moving add phosphoryl glycosyl groups Finally, potential applications complement expand current strategies. Traditional methods removal utilize knockdown knockout strategies interfering RNA, antisense oligonucleotides, CRISPR-associated nucleases), although delivery remain challenge. More recently, endogenous machinery targets. additional reviews triggered how modes action differ predecessors, please see Valeur 2019Valeur Narjes Ottmann C. Plowright A.T. Emerging modes-of-action discovery.Medchemcomm. 2019; 10: 1550-1568Crossref Ding 2020Ding Fei Lu B. concepts degrader technologies.Trends Pharmacol. 41: 464-474Abstract (49) Luh 2020Luh L.M. Scheib U. Juenemann Wortmann L. Brands M. Cromm P.M. Prey proteasome: degradation-A medicinal chemist's perspective.Angew. Int. Ed. Engl. 59: 15448-15466Crossref (37) Scholar. One way ubiquitin E3 ligases, done heterobifunctional termed proteolysis-targeting chimeras (PROTACs) (Sakamoto 2001Sakamoto K.M. Kim K.B. Kumagai Mercurio Deshaies R.J. Protacs: chimeric Skp1-Cullin-F box complex ubiquitination degradation.Proc. 2001; 98: 8554-8559Crossref (810) Pettersson 2019Pettersson PROteolysis TArgeting Chimeras - past, present future.Drug Today Technol. 31: 15-27Crossref (241) Sun 2019Sun X. Gao Yang He Wu Song Tong Rao PROTACs: great opportunities academia industry.Signal. Transduct. Target. Ther. 4: 64Crossref (187) PROTACs formed linking compounds: binds ligase, Von Hippel-Lindau disease tumor suppressor (VHL), cereblon (CRBN), mouse double minute 2 (MDM2). With PROTACs, tagged shuttled proteasome 2A). degraders long-lasting effects since must resynthesized after opposed classic occupancy-driven whereby reactivated upon inhibitor dissociation. Importantly, PROTAC recycled and, therefore, 2001, Deshaies, colleagues reported example methionine aminopeptidase-2 (MetAP2) Since then, variety involved diseases, reviewed extensively (Lai Several challenges still exist testing PROTACs. To improve being targetable considering handful ligases commonly used, over 600 known (Clague 2015Clague M.J. Heride Urbe demographics system.Trends Cell 2015; 25: 417-426Abstract (190) selectivity, enriched (Wang 2017Wang Ma Wang Liu J. Wei W. implications therapies.Cancer Metastasis 36: 683-702Crossref (66) Paiva 2019Paiva Targeted elements design.Curr. Opin. 50: 111-119Crossref Zhang cysteine-reactive electrophiles identify once poorly characterized DCAF16 component ligase (Zhang 2019Zhang Crowley V.M. Wucherpfennig T.G. Dix M.M. Cravatt B.F. Electrophilic nuclear engaging DCAF16.Nat. 737-746Crossref (136) study, Spradlin activity-based profiling product nimbolide reacts cysteine RNF114 (Spradlin 2019Spradlin J.N. Hu Ward C.C. Brittain S.M. Jones M.D. Ou Proudfoot Ornelas Woldegiorgis al.Harnessing anti-cancer degradation.Nat. 747-755Crossref (140) 2019Ward Kleinman J.I. Lee P.S. Chung C.Y.S. Petri Thomas J.R. Tallarico J.A. McKenna al.Covalent ligand uncovers RNF4 recruiter applications.ACS 14: 2430-2440Crossref (100) Both proteomic platforms uncover degradation. expanding space, Cotton antibody-based (AbTAC) recognizes both cell-surface RNF43 membrane-bound programmed death 1 (PD-L1) domains (Cotton 2021Cotton A.D. Nguyen D.P. Gramespacher Seiple I.B. Wells Development immune checkpoint PD-L1.J. 143: 593-598Crossref necessary avenue further studies involves delineation general linker attachment (exit vector). online databases computational tools PROTAC-DB (Weng 2020Weng Shen Cao Dong Q. Li Hou T. PROTAC-DB: database PROTACs.Nucleic Acids Res. 49: D1381-D1387Crossref (29) PRosetTAC (Zaidman 2020Zaidman Prilusky London N. PRosettaC: rosetta based modeling mediated complexes.J. Inf. Model. 60: 4894-4903Crossref (28) emerging, three-component systems difficult. An challenge cell-based assays detecting HiBiT/LgBiT assays; Riching 2018Riching Mahan Corona C.R. McDougall Vasta J.D. Robers M.B. Urh Daniels D.L. Quantitative live-cell kinetic mechanistic mode action.ACS 13: 2758-2770Crossref (105) cooperativity, turnover cells. most off-target focused quantifying alteration levels, when readout should mass spectrometry (Xu 2010Xu Paige J.S. Jaffrey S.R. Global lysine remnant immunoaffinity profiling.Nat. Biotechnol. 2010; 28: 868-873Crossref (367) Udeshi 2013Udeshi N.D. Mertins Svinkina Carr S.A. Large-scale identification spectrometry.Nat. Protoc. 8: 1950-1960Crossref (183) far reaching, several preclinical clinical trial underway orally bioavailable ARV-110 compound targets androgen prostate cancer; Petrylak 2020Petrylak Vogelzang N.J. Garfield M.H. Taylor I. Moore Peck R.A. Burris III, H.A. First-in-human phase I study ARV-110, (AR) patients (pts) metastatic castrate-resistant (mCRPC) following enzalutamide (ENZ) and/or abiraterone (ABI).J. Clin. Oncol. 38: 3500Crossref Further evaluating pharmacokinetic pharmacodynamic properties traditional therapeutics, needed future applications. recruit had substantial success pioneered field small-molecule-targeted mechanism mostly limited exception aforementioned AbTACs. Motivated limitation, lysosomal-targeting (LYTACs) were broaden scope 2B) (Banik 2020Banik Pedram Wisnovsky Ahn Riley N.M. Bertozzi Lysosome-targeting chimaeras proteins.Nature. 584: 291-297Crossref (148) group hypothesized creating receptor, cation-independent mannose-6-phosphate CI-M6PR, enter lysosomal pathway. they identified first-generation stable derivative mannose-6-phosphate, recognized CI-M6PR. They demonstrated glycopeptide polymer linked antibodies successfully exogenously supplied mCherry) EGFR). was multivalency shuttling recycling CI-M6PR back it delivered cargo compartments. much room technology, exploration other targeting receptors instead polymer. effort, co-workers second-generation LYTAC tag, bearing smaller motif, liver-specific asialoglycoprotein (Ahn 2021Ahn Banik C.L. Cochran LYTACs engage degradation.Nat Chem https://doi.org/10.1038/s41589-021-00770-1Crossref folded autophagy greater range substrates, aggregated debris, non-proteinaceous biomolecules. Thus, autophagy-targeting (AUTACs) autophagosome-tethering (ATTECs) potentially robust 2C). major here autophagy, not well understood ubiquitin-proteasomal system. Previous papers antibacterial (xenophagy), cGMP (S-guanylation) autophagosomal clearance. Arimoto tag resembles S-guanylation serve stand-alone (Takahashi 2019Takahashi Moriyama Nakamura Miki Takahashi Sato Akaike Itto-Nakama AUTACs: cargo-specific selective autophagy.Mol. Cell. 76: 797-810.e710Abstract (119) doing so, degraded MetAP2, earliest FKBP12 binders applied technology mitochondrial-fused EGFP-HaloTag protein. destabilized mitochondria, treatment chloroalkane, mitochondrial restored clearing out organelle. AUTAC utilized dysfunctional mitochondria Detroit532 show restoration. autophagy-triggered increases technologies organelles macromolecules, optimize ligands autophagy. cytosol, instance, authors attempted nucleus-residing BRD4, but less successful than cytosolic AUTACs pathway, ATTECs interact directly LC3, autophagosome precursor phagophore formation. side-steps requirement ubiquitin, thus its (Li 2019Li Z. Zhu Sha al.Allele-selective lowering mutant HTT HTT-LC3 compounds.Nature. 575: 203-209Crossref (126) 2020Li ATTEC: approach proteinopathies.Autophagy. 185-187Crossref Moreover, ATTEC AUTACs. ATTEC, Huntington's (HD) stretch glutamines phagophore. Small-molecule found molecular-glue-like interacted HD wild-type vivo models able reverse phenotype affecting disease-relevant glutamine repeats like spinocerebellar ataxia type 3, demonstrating generalizability. Inspired ribonuclease-targeting (RIBOTACs) combine binder ribonuclease nucleases 2D). Alternative methods, oligonucleotides RNAs, extremely poor uptake due size charge (Meyer 2020Meyer Williams Akahori Tanaka Aikawa Childs-Disney J.L. Disney Small structures.Chem. 7167-7199Crossref RIBOTACs medium-sized overcome permeability limitations, serving method First 2018 Costales RIBOTAC L (RNase L) dimerize pri-miR-96, oncogenic (Costales 2018Costales M.G. 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Numerous (the group, PO32?) substrate protein, serine (Ser), threonine (Thr), (Tyr) residues. majority Ser/Thr-specific kinases, Tyr-specific RTKs non-receptor kinases. converse—protein dephosphorylation facilitated phosphatases—is area interest regulation Phosphatases, classically highly promiscuous studied enforce surprisingly degree specificity docking interactions, hydrophobic grooves, certain substrates (Roy Cyert, 2009Roy Cyert Cracking phosphatase code: determine specificity.Sci. Signal. 2009; 2: re9Crossref (134) Sch