Synthesis and Demonstration of the Biological Relevance of sp3 -rich Scaffolds Distantly Related to Natural Product Frameworks.

The productive exploration of chemical space is an enduring challenge in chemical biology and medicinal chemistry. Natural products are biologically relevant, and their frameworks have facilitated chemical tool and drug discovery. A "top-down" synthetic approach is described that enabled a range of complex bridged intermediates to be converted with high step efficiency into 26 diverse sp3 -rich scaffolds. The scaffolds have local natural product-like features, but are only distantly related to specific natural product frameworks. To assess biological relevance, a set of 52 fragments was prepared, and screened by high-throughput crystallography against three targets from two protein families (ATAD2, BRD1 and JMJD2D). In each case, 3D fragment hits were identified that would serve as distinctive starting points for ligand discovery. This demonstrates that frameworks that are distantly related to natural products can facilitate discovery of new biologically relevant regions within chemical space.

Catalytic in vivo protein knockdown by small-molecule PROTACs.

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.

Sigmatropic Rearrangement of Vinyl Aziridines: Expedient Synthesis of Cyclic Sulfoximines from Chiral Sulfinimines.

A novel rearrangement of 2-vinyl aziridine 2-carboxylates to unusual chiral cyclic sulfoximines is described herein. The method allows the synthesis of substituted cyclic sulfoximines in high yields with complete stereocontrol, and tolerates a wide substrate scope. A one-pot process starting directly from sulfinimines provides access to complex chiral sulfoximines in only two steps from commercially available aldehydes. A mechanistic hypothesis and synthetic application in the formal synthesis of trachelanthamidine, by transformation of a cyclic sulfoximine into a pyrroline, is also disclosed.

A unified lead-oriented synthesis of over fifty molecular scaffolds.

Controlling the properties of lead molecules is critical in drug discovery, but sourcing large numbers of lead-like compounds for screening collections is a major challenge. A unified synthetic approach is described that enabled the synthesis of 52 diverse lead-like molecular scaffolds from a minimal set of 13 precursors. The divergent approach exploited a suite of robust, functional group-tolerant transformations. Crucially, after derivatisation, these scaffolds would target significant lead-like chemical space, and complement commercially-available compounds.

Lead-oriented synthesis: a new opportunity for synthetic chemistry.

The pharmaceutical industry remains solely reliant on synthetic chemistry methodology to prepare compounds for small-molecule drug discovery programmes. The importance of the physicochemical properties of these molecules in determining their success in drug development is now well understood but we present here data suggesting that much synthetic methodology is unintentionally predisposed to producing molecules with poorer drug-like properties. This bias may have ramifications to the early hit- and lead-finding phases of the drug discovery process when larger numbers of compounds from array techniques are prepared. To address this issue we describe for the first time the concept of lead-oriented synthesis and the opportunity for its adoption to increase the range and quality of molecules used to develop new medicines.

Stereoselective aza-Darzens reactions of tert-butanesulfinimines: convenient access to chiral aziridines.

Stereoselective synthesis of 2,3-di- and 2,2',3-tri-substituted aziridines in good yields and excellent diastereoselectivities are achieved through aza-Darzens reactions of a range of tert-butanesulfinyl aldimines and ketimines with ethyl bromoacetate.

The rise and rise of protein degradation: Opportunities and challenges ahead.

The transformational mechanism of action underpinning targeted protein degradation strategies, including proteolysis-targeting chimeras (PROTACs), gives potential for potent in vivo pharmacology and has allowed projects to move rapidly to the clinic. Despite this remarkable progress, there remain many opportunities to improve current, first-generation approaches even further. Our expanding knowledge will allow discovery of new degrading mechanisms with potential to address several limitations of current approaches, including improving scope and efficiency of degradation, improving drug-like properties of degraders, and reducing potential for the emergence of acquired resistance. Here, we discuss potential routes to realize these advances to expand TPD utility even further.

Optimization of a Series of RIPK2 PROTACs.

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.

Structural Insights into PROTAC-Mediated Degradation of Bcl-xL.

The Bcl-2 family of proteins, such as Bcl-xL and Bcl-2, play key roles in cancer cell survival. Structural studies of Bcl-xL formed the foundation for the development of the first Bcl-2 family inhibitors and FDA approved drugs. Recently, Proteolysis Targeting Chimeras (PROTACs) that degrade Bcl-xL have been proposed as a therapeutic modality with the potential to enhance potency and reduce toxicity versus antagonists. However, no ternary complex structures of Bcl-xL with a PROTAC and an E3 ligase have been successfully determined to guide this approach. Herein, we report the design, characterization, and X-ray structure of a VHL E3 ligase-recruiting Bcl-xL PROTAC degrader. The 1.9 Å heterotetrameric structure, composed of (ElonginB:ElonginC:VHL):PROTAC:Bcl-xL, reveals an extensive network of neo-interactions, between the E3 ligase and the target protein, and between noncognate parts of the PROTAC and partner proteins. This work illustrates the challenges associated with the rational design of bifunctional molecules where interactions involve composite interfaces.

Extended pharmacodynamic responses observed upon PROTAC-mediated degradation of RIPK2.

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.

Protac-Induced Protein Degradation in Drug Discovery: Breaking the Rules or Just Making New Ones?

Targeted protein degradation, using bifunctional small molecules (Protacs) to remove specific proteins from within cells, has emerged as a novel drug discovery strategy with the potential to offer therapeutic interventions not achievable with existing approaches. In this Perspective, the brief history of the field is surveyed from a drug discovery perspective with a focus on the key advances in knowledge which have led to the definition and exemplification of protein degradation concepts and their resulting applications to medicine discovery. The approach has the potential to bring disruptive change to drug discovery; the many potential advantages and outstanding challenges which lie ahead of this technology are discussed.

Organic synthesis provides opportunities to transform drug discovery.

Despite decades of ground-breaking research in academia, organic synthesis is still a rate-limiting factor in drug-discovery projects. Here we present some current challenges in synthetic organic chemistry from the perspective of the pharmaceutical industry and highlight problematic steps that, if overcome, would find extensive application in the discovery of transformational medicines. Significant synthesis challenges arise from the fact that drug molecules typically contain amines and N-heterocycles, as well as unprotected polar groups. There is also a need for new reactions that enable non-traditional disconnections, more C-H bond activation and late-stage functionalization, as well as stereoselectively substituted aliphatic heterocyclic ring synthesis, C-X or C-C bond formation. We also emphasize that syntheses compatible with biomacromolecules will find increasing use, while new technologies such as machine-assisted approaches and artificial intelligence for synthesis planning have the potential to dramatically accelerate the drug-discovery process. We believe that increasing collaboration between academic and industrial chemists is crucial to address the challenges outlined here.

Modulating PCAF/GCN5 Immune Cell Function through a PROTAC Approach.

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.

Tau therapeutic strategies for the treatment of Alzheimer's disease.

The two classical pathological hallmarks of Alzheimer's disease are deposits of aggregated beta-amyloid (Abeta) peptide and neurofibrillary tangles composed of hyperphosphorylated tau protein. In addition to Abeta pathology, an invariant trait of Alzheimer's disease, disruption of tau processing is a necessary event in the neurotoxic cascade which eventually leads to neuronal death and subsequent dementia. Tau is a neuronal, microtubule-bound protein which becomes hyperphosphorylated as a result of an imbalance of the kinase and phosphatase activities which normally tightly regulate its phosphorylation. In addition to this pathogenic hyperphosphorylation, tau dissociates from microtubules and self-aggregates to form insoluble oligomers which progress to the macroscopic tangles evident in post mortem Alzheimer's disease tissue. Subsequent toxicity may ensue either as a direct toxic effect of free tau oligomers or as a result of altered microtubule-dependent processes. In order to intervene pharmacologically in this disease process, much effort has been expended in order to identify and inhibit the kinases responsible for pathogenic hyperphosphorylation and many candidate kinases have been investigated including glycogen synthase kinase (GSK-3), cyclin-dependant kinase-5 (Cdk-5), MAPK family members (extracellular signal-regulated kinases 1 and 2 [Erk-1 and 2], MEK [MAP kinase kinase], c-Jun NH(2)-terminal kinases (JNKs) and p38), casein kinase, calcium calmodulin-dependant kinase II (CaMK-II), microtubule affinity regulating kinase (MARK), protein kinase A (PKA/cAMP-dependant protein kinase) and others. Focus has also fallen upon the role of the phosphatases responsible for dephosphorylation of tau. This review will describe the tau-related etiology of Alzheimer's disease and other tauopathies as well as the therapeutic strategies to inhibit the hyperphosphorylation of tau.

A divergent synthetic approach to diverse molecular scaffolds: assessment of lead-likeness using LLAMA, an open-access computational tool.

Complementary cyclisation reactions of hex-2-ene-1,6-diamine derivatives were exploited in the synthesis of alternative molecular scaffolds. The value of the synthetic approach was analysed using LLAMA, an open-access computational tool for assessing the lead-likeness and novelty of molecular scaffolds.

Structurally Diverse Mitochondrial Branched Chain Aminotransferase (BCATm) Leads with Varying Binding Modes Identified by Fragment Screening.

Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility.

Gamma-secretase as a therapeutic target for the treatment of Alzheimer's disease.

An effective, disease-modifying treatment of Alzheimer's disease (AD) remains one of the most significant unmet needs in modern medicine. As a result of the extensive research in the area, the mechanisms underlying the disease are now much better understood than at any time before. A significant amount of evidence points to the central role of beta-amyloid (Abeta) peptide-mediated toxicity in the disease etiology and strategies to remove this species from the central nervous system (CNS) have been actively pursued. The enzyme responsible for the final step in Abeta synthesis, gamma-secretase, has emerged as an attractive drug target and intensive research has transformed this enzyme from shadowy beginnings into a well characterised member of a new family of intramembrane-cleaving aspartyl proteases. Many inhibitors across diverse structural classes have been discovered and have demonstrated a lowering of central Abeta levels in preclinical models of AD. It has also become increasingly evident more recently that gamma-secretase also mediates a range of cleavages of alternative transmembrane peptides most notably the Notch receptor and the functional consequences of this activity have attracted much attention. The ultimate therapeutic benefit of gamma-secretase inhibitors and the effect of alternative, mechanism-based activities can only be judged when clinical data is forthcoming. In this review we describe the literature regarding the discovery of the nature of gamma-secretase, the development of small molecule inhibitors and their in vivo profiles.

A systematic approach to diverse, lead-like scaffolds from α,α-disubstituted amino acids.

A powerful strategy for the efficient lead-oriented synthesis of novel molecular scaffolds is demonstrated. Twenty two scaffolds were prepared from just four α-amino acid-derived building blocks and a toolkit of six connective reactions. Importantly, each individual scaffold has the ability to specifically target lead-like chemical space.

The Discovery of in Vivo Active Mitochondrial Branched-Chain Aminotransferase (BCATm) Inhibitors by Hybridizing Fragment and HTS Hits.

The hybridization of hits, identified by complementary fragment and high throughput screens, enabled the discovery of the first series of potent inhibitors of mitochondrial branched-chain aminotransferase (BCATm) based on a 2-benzylamino-pyrazolo[1,5-a]pyrimidinone-3-carbonitrile template. Structure-guided growth enabled rapid optimization of potency with maintenance of ligand efficiency, while the focus on physicochemical properties delivered compounds with excellent pharmacokinetic exposure that enabled a proof of concept experiment in mice. Oral administration of 2-((4-chloro-2,6-difluorobenzyl)amino)-7-oxo-5-propyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-3-carbonitrile 61 significantly raised the circulating levels of the branched-chain amino acids leucine, isoleucine, and valine in this acute study.

gamma-Secretase as a target for drug intervention in Alzheimer's disease.

gamma-Secretase is a critical enzyme involved in the production of amyloid-beta (Abeta) peptide, one of the main pathological hallmarks of Alzheimer's disease. gamma-Secretase cleaves the beta-amyloid precursor protein (betaAPP) at a position predicted to be within the membrane. In addition to betaAPP, gamma-secretase cleaves a range of other substrates. Thus, a key question in the development of gamma-secretase inhibitors for preventing Abeta production is whether undesired mechanism-based side effects may result from inhibition of cleavage of other substrates, and if so whether a suitable window exists to reduce brain Abeta. In this review, progress in the development of small-molecule inhibitors will be described, and potential toxicity issues associated with the development of gamma-secretase inhibitors discussed.