ABSTRACT
Targeted protein degraders such as PROTACs and molecular glues are a rapidly emerging therapeutic modality within industry and academia. Degraders possess unique mechanisms of action that lead to the removal of specific proteins by co-opting the cell's natural degradation mechanisms via induced proximity. Their optimisation thus far has often been largely empirical, requiring the synthesis and screening of a large number of analogues. In addition, the synthesis and development of degraders is often challenging, leading to lengthy optimisation campaigns to deliver candidate-quality compounds. This review highlights how the synthesis of degraders has evolved in recent years, in particular focusing on means of applying high-throughput chemistry and screening approaches to expedite these timelines, which we anticipate to be valuable in shaping the future of degrader optimisation campaigns.
Subject(s)
Combinatorial Chemistry Techniques , High-Throughput Screening Assays , Proteins/chemistry , Proteins/metabolism , Proteolysis , Small Molecule Libraries/chemistry , Small Molecule Libraries/chemical synthesisABSTRACT
Macrocycles and medium-sized rings are important in many scientific fields and technologies but are hard to make using current methods, especially on a large scale. Outlined herein is a strategy by which functionalized macrocycles and medium-sized rings can be prepared using cyclization/ring expansion (CRE) cascade reactions, without resorting to high dilution conditions. CRE cascade reactions are designed to operate exclusively via kinetically favorable 5-7-membered ring cyclization steps; this means that the problems typically associated with classical end-to-end macrocyclization reactions are avoided. A modular synthetic approach has been developed to facilitate the simple assembly of the requisite linear precursors, which can then be converted into an extremely broad range of functionalized macrocycles and medium-sized rings using one of nine CRE protocols.
ABSTRACT
Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that co-opt the cell's natural proteasomal degradation mechanisms to degrade undesired proteins. A challenge associated with PROTACs is the time and resource-intensive optimization; thus, the development of high-throughput platforms for their synthesis and biological evaluation is required. In this study, we establish an ultra-high-throughput experimentation (ultraHTE) platform for PROTAC synthesis, followed by direct addition of the crude reaction mixtures to cellular degradation assays without any purification. This 'direct-to-biology' (D2B) approach was validated and then exemplified in a medicinal chemistry campaign to identify novel BRD4 PROTACs. Using the D2B platform, the synthesis of 650 PROTACs was carried out in a 1536-well plate, and subsequent biological evaluation was performed by a single scientist in less than 1 month. Due to its ability to hugely accelerate the optimization of new degraders, we anticipate our platform will transform the synthesis and testing of PROTACs.
Subject(s)
Nuclear Proteins , Proteolysis Targeting Chimera , Transcription Factors , Biological Assay , Biology , Proteolysis , Ubiquitin-Protein LigasesABSTRACT
Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is an important kinase of the innate immune system. Herein, we describe the optimization of a series of RIPK2 PROTACs which recruit members of the inhibitor of apoptosis (IAP) family of E3 ligases. Our PROTAC optimization strategy focused on reducing the lipophilicity of the early lead which resulted in the identification of analogues with improved solubility and increased human and rat microsomal stability. We identified a range of IAP binders that were successfully incorporated into potent RIPK2 PROTACs with attractive pharmacokinetic profiles. Compound 20 possessed the best overall profile with good solubility, potent degradation of RIPK2, and associated inhibition of TNFα release. A proof-of-concept study utilizing a slow release matrix demonstrated the feasibility of a long-acting parenteral formulation with >1 month duration. This represents an attractive alternative dosing paradigm to oral delivery, especially for chronic diseases where compliance can be challenging.
Subject(s)
Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Animals , Drug Design , Gene Expression Regulation/drug effects , Half-Life , Humans , Male , Molecular Structure , Rats , Rats, Sprague-Dawley , Rats, Wistar , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , THP-1 CellsABSTRACT
Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Drug Design , Inflammation/prevention & control , Leukocytes, Mononuclear/drug effects , Proteasome Endopeptidase Complex/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/pharmacokinetics , Colitis, Ulcerative/drug therapy , Colitis, Ulcerative/enzymology , Crohn Disease/drug therapy , Crohn Disease/enzymology , Cytokines/metabolism , Dose-Response Relationship, Drug , Enzyme Stability , Female , Humans , Inflammation/enzymology , Inflammation/immunology , Inflammation Mediators/metabolism , Injections, Intravenous , Leukocytes, Mononuclear/enzymology , Male , Proteolysis , Rats, Sprague-Dawley , Rats, Wistar , THP-1 Cells , Tissue Culture Techniques , UbiquitinationABSTRACT
P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are closely related epigenetic proteins, each containing an acetyltransferase domain and a bromodomain. Consistent with reported roles for these proteins in immune function, we find that PCAF-deficient macrophages exhibit a markedly reduced ability to produce cytokines upon stimulation with lipopolysaccharide (LPS). Investigating the potential to target this pathway pharmacologically, we show that chemical inhibition of the PCAF/GCN5 bromodomains is insufficient to recapitulate the diminished inflammatory response of PCAF-deficient immune cells. However, by generating the first PCAF/GCN5 proteolysis targeting chimera (PROTAC), we identify small molecules able to degrade PCAF/GCN5 and to potently modulate the expression of multiple inflammatory mediators in LPS-stimulated macrophages and dendritic cells. Our data illustrate the power of the PROTAC approach in the context of multidomain proteins, revealing a novel anti-inflammatory therapeutic opportunity for targeting PCAF/GCN5.
Subject(s)
Benzoates/pharmacology , Piperidines/pharmacology , Pyridazines/pharmacology , p300-CBP Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing , Animals , Benzoates/chemical synthesis , Benzoates/chemistry , Cell Differentiation/drug effects , Cytokines/metabolism , Dendritic Cells/metabolism , Humans , Inflammation/chemically induced , Inflammation/metabolism , Lipopolysaccharides , Macrophages/metabolism , Mice , Monocytes/metabolism , Peptide Hydrolases/metabolism , Piperidines/chemical synthesis , Piperidines/chemistry , Protein Domains , Proteolysis , Pyridazines/chemical synthesis , Pyridazines/chemistry , Stereoisomerism , Ubiquitin-Protein Ligases , p300-CBP Transcription Factors/chemistryABSTRACT
A novel 4-aminoindazole sulfonamide hit (13) was identified as a human CCR4 antagonists from testing a focussed library of compounds in the primary GTPγS assay. Replacing the indazole core with a pyrazolopyrimidine, and introduction of a methoxy group adjacent to the sulfonamide substituent, resulted in the identification of pyrazolopyrimidine 37a, which exhibited good binding affinity in the GTPγS assay (pIC50=7.2), low lipophilicity (clogP=2.2, chromlogD7.4=2.4), high LE (0.41), high solubility (CLND solubility ≥581µM), and an excellent PK profile in both the rat (F=62%) and the dog (F=100%). Further SAR investigation of the pyrazolopyrimidine suggested that substitution at N1 is tolerated, providing a suitable vector to modulate the properties, and increase the potency in a lead optimisation campaign.
Subject(s)
Receptors, CCR4/antagonists & inhibitors , Sulfonamides/pharmacology , Animals , Dogs , Dose-Response Relationship, Drug , Humans , Male , Molecular Structure , Rats , Rats, Sprague-Dawley , Rats, Wistar , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistryABSTRACT
A number of potent 2,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine CCR4 antagonists binding to the extracellular allosteric site were synthesised. (R)-N-(2,4-Dichlorobenzyl)-2-(2-(pyrrolidin-2-ylmethyl)-2,8-diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine (R)-(18a) has high affinity in both the [(125)I]-TARC binding assay with a pKi of 8.8, and the [(35)S]-GTPγS functional assay with a pIC50 of 8.1, and high activity in the human whole blood actin polymerisation assay (pA2 = 6.7). The most potent antagonists were also investigated for their ability to induce endocytosis of CCR4 and were found to internalise about 60% of the cell surface receptors, a property which is not commonly shared by small molecule antagonists of chemokine receptors.
Subject(s)
Endocytosis/drug effects , Pyrimidines/pharmacology , Receptors, CCR4/antagonists & inhibitors , Spiro Compounds/pharmacology , Dose-Response Relationship, Drug , Humans , Molecular Structure , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Receptors, CCR4/metabolism , Spiro Compounds/chemical synthesis , Spiro Compounds/chemistry , Structure-Activity RelationshipABSTRACT
The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Neoplasm Proteins/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinase 2/antagonists & inhibitors , Receptors, Estrogen/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Animals , Binding Sites , Biocatalysis , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Humans , MCF-7 Cells , Mice , Models, Molecular , Molecular Targeted Therapy , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasm Transplantation , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Proteolysis , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitination , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , ERRalpha Estrogen-Related ReceptorABSTRACT
Small molecule-induced protein degradation is an attractive strategy for the development of chemical probes. One method for inducing targeted protein degradation involves the use of PROTACs, heterobifunctional molecules that can recruit specific E3 ligases to a desired protein of interest. PROTACs have been successfully used to degrade numerous proteins in cells, but the peptidic E3 ligase ligands used in previous PROTACs have hindered their development into more mature chemical probes or therapeutics. We report the design of a novel class of PROTACs that incorporate small molecule VHL ligands to successfully degrade HaloTag7 fusion proteins. These HaloPROTACs will inspire the development of future PROTACs with more drug-like properties. Additionally, these HaloPROTACs are useful chemical genetic tools, due to their ability to chemically knock down widely used HaloTag7 fusion proteins in a general fashion.
Subject(s)
Proteolysis , Recombinant Fusion Proteins/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Ubiquitin-Protein Ligases/metabolism , HEK293 Cells , Humans , Ligands , Protein BindingABSTRACT
A knowledge-based library of 2,3-dichlorophenylsulfonyl derivatives of commercially available aryl amines was synthesised and screened as human CCR4 antagonists, in order to identify a suitable hit for the start of a lead-optimisation programme. Hits were required to be more potent than an existing indazole series, have better physicochemical properties (clogP <3.5, chrom logD7.4 <5.3 and CLND solubility >116 µg/mL), and be stable to acid and light. The benzimidazol-2-one core was identified as a hit suitable for further investigation. Substitution at N1 with small alkyl groups was tolerated; however, these analogues were inactive in the whole blood assay (pA2 <5). Azabenzimidazolone analogues were all found to be active, with compound 38 exhibiting whole blood activity of 6.1, low molecular weight (389) and chrom logD7.4 (2.4), high LE (0.43), and solubility (152 µg/mL). In addition, 38 had human serum albumin binding of around 93% and met all the criteria for progression to lead optimisation.
Subject(s)
Benzimidazoles/chemistry , Receptors, CCR4/antagonists & inhibitors , Sulfonamides/chemistry , Aza Compounds/chemistry , Humans , Indazoles/chemistry , Protein Binding , Receptors, CCR4/metabolism , Serum Albumin/chemistry , Serum Albumin/metabolism , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/metabolismABSTRACT
A knowledge-based library of aryl 2,3-dichlorophenylsulfonamides was synthesised and screened as human CCR4 antagonists, in order to identify a suitable hit for the start of a lead-optimisation programme. X-ray diffraction studies were used to identify the pyrazole ring as a moiety that could bring about intramolecular hydrogen bonding with the sulfonamide NH and provide a clip or orthogonal conformation that was believed to be the preferred active conformation. Replacement of the core phenyl ring with a pyridine, and replacement of the 2,3-dichlorobenzenesulfonamide with 5-chlorothiophenesulfonamide provided compound 33 which has excellent physicochemical properties and represents a good starting point for a lead optimisation programme. Electronic structure calculations indicated that the preference for the clip or orthogonal conformation found in the small molecule crystal structures of 7 and 14 was in agreement with the order of potency in the biological assay.
Subject(s)
Pyrazoles/chemistry , Receptors, CCR4/antagonists & inhibitors , Sulfonamides/chemistry , Sulfonamides/pharmacology , Allosteric Regulation , Models, Molecular , Molecular Conformation , Structure-Activity RelationshipABSTRACT
A series of indazole arylsulfonamides were synthesized and examined as human CCR4 antagonists. Methoxy- or hydroxyl-containing groups were the more potent indazole C4 substituents. Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups possessing an α-amino-3-[(methylamino)acyl]-group were the most potent N1-substituents. Strongly basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good oral absorption; however, they also had high clearance. The most potent compound with high absorption in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular interaction in the active conformation.