Small-molecule factor B inhibitor for the treatment of complement-mediated diseases.

Dysregulation of the alternative complement pathway (AP) predisposes individuals to a number of diseases including paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Moreover, glomerular Ig deposits can lead to complement-driven nephropathies. Here we describe the discovery of a highly potent, reversible, and selective small-molecule inhibitor of factor B, a serine protease that drives the central amplification loop of the AP. Oral administration of the inhibitor prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats. In addition, inhibition of factor B prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes. These data demonstrate the potential therapeutic value of using a factor B inhibitor for systemic treatment of complement-mediated diseases and provide a basis for its clinical development.

Glucagon Like Peptide 1 Receptor Agonists for Targeted Delivery of Antisense Oligonucleotides to Pancreatic Beta Cell.

The extra hepatic delivery of antisense oligonucleotides (ASOs) remains a challenge and hampers the widespread application of this powerful class of therapeutic agents. In that regard, pancreatic beta cells are a particularly attractive but challenging cell type because of their pivotal role in diabetes and the fact that they are refractory to uptake of unconjugated ASOs. To circumvent this, we have expanded our understanding of the structure activity relationship of ASOs conjugated to Glucagon Like Peptide 1 Receptor (GLP1R) agonist peptide ligands. We demonstrate the key role of the linker chemistry and its optimization to design maleimide based conjugates with improved in vivo efficacy. In addition, truncation studies and scoping of a diverse set of GLP1R agonists proved fruitful to identify additional targeting ligands efficacious in vivo including native hGLP1(7-36)NH2. Variation of the carrier peptide also shed some light on the dramatic impact of subtle sequence differences on the corresponding ASO conjugate performance in vivo, an area which clearly warrant further investigations. We have confirmed the remarkable potential of GLP1R agonist conjugation for the delivery of ASOs to pancreatic beta cell by effectively knocking down islet amyloid polypeptide (IAPP) mRNA, a potential proapoptotic target, in mice.

Macrocyclic Modalities Combining Peptide Epitopes and Natural Product Fragments.

"Hot loop" protein segments have variable structure and conformation and contribute crucially to protein-protein interactions. We describe a new hot loop mimicking modality, termed PepNats, in which natural product (NP)-inspired structures are incorporated as conformation-determining and -restricting structural elements into macrocyclic hot loop-derived peptides. Macrocyclic PepNats representing hot loops of inducible nitric oxide synthase (iNOS) and human agouti-related protein (AGRP) were synthesized on solid support employing macrocyclization by imine formation and subsequent stereoselective 1,3-dipolar cycloaddition as key steps. PepNats derived from the iNOS DINNN hot loop and the AGRP RFF hot spot sequence yielded novel and potent ligands of the SPRY domain-containing SOCS box protein 2 (SPSB2) that binds to iNOS, and selective ligands for AGRP-binding melanocortin (MC) receptors. NP-inspired fragment absolute configuration determines the conformation of the peptide part responsible for binding. These results demonstrate that combination of NP-inspired scaffolds with peptidic epitopes enables identification of novel hot loop mimics with conformationally constrained and biologically relevant structure.

Late-Stage Functionalization of Histidine in Unprotected Peptides.

The late-stage functionalization (LSF) of peptides represents a valuable strategy for the design of potent peptide pharmaceuticals by enabling rapid exploration of chemical diversity and offering novel opportunities for peptide conjugation. While the C(sp2 )-H activation of tryptophan (Trp) is well documented, the resurgence of radical chemistry is opening new avenues for the C-H functionalization of other aromatic side-chains. Herein, we report the first example of LSF at C2 of histidine (His) utilizing a broad scope of aliphatic sulfinate salts as radical precursors. In this work, the exquisite selectivity for histidine functionalization was demonstrated through the alkylation of complex unprotected peptides in aqueous media. Finally, this methodology was extended for the installation of a ketone handle, providing an unprecedented anchor for selective oxime/hydrazone conjugation at histidine.

Structure-based design and synthesis of macrocyclic human rhinovirus 3C protease inhibitors.

The design and synthesis of macrocyclic inhibitors of human rhinovirus 3C protease is described. A macrocyclic linkage of the P1 and P3 residues, and the subsequent structure-based optimization of the macrocycle conformation and size led to the identification of a potent biochemical inhibitor 10 with sub-micromolar antiviral activity.

New Modalities for Challenging Targets in Drug Discovery.

Our ever-increasing understanding of biological systems is providing a range of exciting novel biological targets, whose modulation may enable novel therapeutic options for many diseases. These targets include protein-protein and protein-nucleic acid interactions, which are, however, often refractory to classical small-molecule approaches. Other types of molecules, or modalities, are therefore required to address these targets, which has led several academic research groups and pharmaceutical companies to increasingly use the concept of so-called "new modalities". This Review defines for the first time the scope of this term, which includes novel peptidic scaffolds, oligonucleotides, hybrids, molecular conjugates, as well as new uses of classical small molecules. We provide the most representative examples of these modalities to target large binding surface areas such as those found in protein-protein interactions and for biological processes at the center of cell regulation.

Structure-based design of 7-azaindole-pyrrolidine amides as inhibitors of 11ß-hydroxysteroid dehydrogenase type I.

Indole-pyrrolidines were identified as inhibitors of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) by high-throughput screening. Optimisation of the initial hit through structure-based design led to 7-azaindole-derivatives, with the best analogues displaying single digit nanomolar IC(50) potency. The modeling hypotheses were confirmed by solving the X-ray co-crystal structure of one of the lead compounds. These compounds were selective against 11ß-hydroxysteroid dehydrogenase type 2 (selectivity ratio >200) and exhibited good inhibition of 11ß-HSD1 (IC(50)<1µM) in a cellular model (3T3L1 adipocytes).

A protein tertiary structure mimetic modulator of the Hippo signalling pathway.

Transcription factors are key protein effectors in the regulation of gene transcription, and in many cases their activity is regulated via a complex network of protein-protein interactions (PPI). The chemical modulation of transcription factor activity is a long-standing goal in drug discovery but hampered by the difficulties associated with the targeting of PPIs, in particular when extended and flat protein interfaces are involved. Peptidomimetics have been applied to inhibit PPIs, however with variable success, as for certain interfaces the mimicry of a single secondary structure element is insufficient to obtain high binding affinities. Here, we describe the design and characterization of a stabilized protein tertiary structure that acts as an inhibitor of the interaction between the transcription factor TEAD and its co-repressor VGL4, both playing a central role in the Hippo signalling pathway. Modification of the inhibitor with a cell-penetrating entity yielded a cell-permeable proteomimetic that activates cell proliferation via regulation of the Hippo pathway, highlighting the potential of protein tertiary structure mimetics as an emerging class of PPI modulators.

Discovery of 4-((2S,4S)-4-Ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic Acid (LNP023), a Factor B Inhibitor Specifically Designed To Be Applicable to Treating a Diverse Array of Complement Mediated Diseases.

The alternative pathway (AP) of the complement system is a key contributor to the pathogenesis of several human diseases including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and various glomerular diseases. The serine protease factor B (FB) is a key node in the AP and is integral to the formation of C3 and C5 convertase. Despite the prominent role of FB in the AP, selective orally bioavailable inhibitors, beyond our own efforts, have not been reported previously. Herein we describe in more detail our efforts to identify FB inhibitors by high-throughput screening (HTS) and leveraging insights from several X-ray cocrystal structures during optimization efforts. This work culminated in the discovery of LNP023 (41), which is currently being evaluated clinically in several diverse AP mediated indications.

Discovery and structure-activity relationships of pentanedioic acid diamides as potent inhibitors of 11beta-hydroxysteroid dehydrogenase type I.

Benzylamides of pentanedioic acid were identified as inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) by high-throughput screening. Optimisation to 2-adamantyl amides yielded inhibitors with single digit nanomolar IC(50)s on the 11beta-HSD1 human isoform. The hydroxy adamantyl amide lead compound was selective against 11beta-hydroxysteroid dehydrogenase type 2 (selectivity ratio >1000) and displayed good inhibition of 11beta-HSD1 (IC(50)<0.1microM) in a cellular model (3T3L1 adipocytes).

Discovery and structure-guided drug design of inhibitors of 11beta-hydroxysteroid-dehydrogenase type I based on a spiro-carboxamide scaffold.

Spiro-carboxamides were identified as inhibitors of 11beta-hydroxysteroid-dehydrogenase type 1 by high-throughput screening. Structure-based drug design was used to optimise the initial hit yielding a sub-nanomolar IC(50) inhibitor (0.5nM) on human 11beta-HSD1 with a high binding efficiency index (BEI of 32.7) which was selective against human 11beta-HSD2 (selectivity ratio>200000).

Emerging modes-of-action in drug discovery.

An increasing focus on complex biology to cure diseases rather than merely treat symptoms has transformed how drug discovery can be approached. Instead of activating or blocking protein function, a growing repertoire of drug modalities can be leveraged or engineered to hijack cellular processes, such as translational regulation or degradation mechanisms. Drug hunters can therefore access a wider arsenal of modes-of-action to modulate biological processes and this review summarises these emerging strategies by highlighting the most representative examples of these approaches.

Analysis of chemical shift changes reveals the binding modes of isoindolinone inhibitors of the MDM2-p53 interaction.

In this study we present a method for defining the binding modes of a set of structurally related isoindolinone inhibitors of the MDM2-p53 interaction. This approach derives the location and orientation of isoindolinone binding, based on an analysis of the patterns of magnitude and direction of chemical shift perturbations for a series of inhibitors of the MDM2-p53 interaction. The MDM2-p53 complex is an attractive target for therapeutic intervention in cancer cells with intact tumor suppressor p53, as it offers the possibility of releasing p53 by blocking the MDM2-p53 binding site with a small molecule antagonist to promote apoptosis. Isoindolinones are a novel class of MDM2-antagonists of moderate affinity, which still require the development of more potent candidates for clinical applications. As the applicability of conventional structural methods to this system is limited by a number of fundamental factors, the exploitation of the information contained in chemical shift perturbations has offered a useful route to obtaining structural information to guide the development of more potent compounds. For a set of 12 structurally related isoindolinones, the data suggests 4 different orientations of binding, caused by subtle changes in the chemical structure of the inhibitors.

New Modalities, Technologies, and Partnerships in Probe and Lead Generation: Enabling a Mode-of-Action Centric Paradigm.

With the rise of novel biology and high potential target identification technologies originating from advances in genomics, medicinal chemists are progressively facing targets of increasing complexity and often unprecedented. Novel hit finding technologies, combined with a wider choice of drug modalities, has resulted in a unique repertoire of options to address these challenging targets and to identify suitable starting points for optimization. Furthermore, innovative solutions originating from a range of academic groups and biotech companies require new types of collaborative models to leverage and integrate them in the drug discovery process. This perspective provides a guide for medicinal chemists covering contemporary probe and lead generation approaches and discusses the strengths and limitations of each strategy. Moreover, the expansion of strategies to modulate proteins creates the opportunity of a modality-agnostic and mode-of-action centric hit finding paradigm.

Inhibition of low-density lipoprotein receptor degradation with a cyclic peptide that disrupts the homodimerization of IDOL E3 ubiquitin ligase.

Cellular uptake of circulating cholesterol occurs via the low density lipoprotein receptor (LDLR). The E3 ubiquitin ligase IDOL is a mediator of LDLR degradation, with IDOL homodimerization thought to be required for its activity. To probe the possibility of modulating LDLR levels with an inhibitor of IDOL homodimerization, we screened a SICLOPPS library of 3.2 million cyclic peptides for compounds that disrupt this protein-protein interaction. We identified cyclo-CFFLYT as the lead inhibitor, and improved its activity through the incorporation of non-natural amino acids. The activity of the optimized cyclic peptide was assessed in hepatic cells, with a dose-dependent increase in LDLR levels observed in the presence of our IDOL homodimerization inhibitor.

Directing evolution: the next revolution in drug discovery?

The strong biological rationale to pursue challenging drug targets such as protein-protein interactions has stimulated the development of novel screening strategies, such as DNA-encoded libraries, to allow broader areas of chemical space to be searched. There has also been renewed interest in screening natural products, which are the result of evolutionary selection for a function, such as interference with a key signalling pathway of a competing organism. However, recent advances in several areas, such as understanding of the biosynthetic pathways for natural products, synthetic biology and the development of biosensors to detect target molecules, are now providing new opportunities to directly harness evolutionary pressure to identify and optimize compounds with desired bioactivities. Here, we describe innovations in the key components of such strategies and highlight pioneering examples that indicate the potential of the directed-evolution concept. We also discuss the scientific gaps and challenges that remain to be addressed to realize this potential more broadly in drug discovery.

Targeted delivery for regenerative medicines: an untapped opportunity for drug conjugates.

Regenerative approaches are promising avenues to effectively cure diseases rather than merely treating symptoms, but are associated with concerns around proliferation in other organs. Given that targeted delivery holds the promise of delivering a drug precisely to its desired site of action, usually with the prospect of increasing the therapeutic index, it can be considered as an essential enabler of regenerative medicines. Although significant progress has been made predominantly in oncology for the delivery of cytotoxic drugs using antibody-drug conjugates (ADCs), the physiological conditions and safety requirements for regenerative medicines are very different. Drug conjugates need to be approached differently and, we herein suggest using a broader range of homing modalities and a specific framework to develop safe linkers.

Therapeutic Potential of Foldamers: From Chemical Biology Tools To Drug Candidates?

Over the past decade, foldamers have progressively emerged as useful architectures to mimic secondary structures of proteins. Peptidic foldamers, consisting of various amino acid based backbones, have been the most studied from a therapeutic perspective, while polyaromatic foldamers have barely evolved from their nascency and remain perplexing for medicinal chemists due to their poor drug-like nature. Despite these limitations, this compound class may still offer opportunities to study challenging targets or provide chemical biology tools. The potential of foldamer drug candidates reaching the clinic is still a stretch. Nevertheless, advances in the field have demonstrated their potential for the discovery of next generation therapeutics. In this perspective, the current knowledge of foldamers is reviewed in a drug discovery context. Recent advances in the early phases of drug discovery including hit finding, target validation, and optimization and molecular modeling are discussed. In addition, challenges and focus areas are debated and gaps highlighted.

PS-IIDQ: an efficient polymer-supported amide coupling reagent.

Polystyrene-IIDQ is a polymer-supported coupling reagent that couples carboxylic acids to amines in good yields and high purity without the requirement of a pre-activation step. Importantly the order of addition of the amine, acid or coupling agent makes no difference to the efficiency of the coupling reaction and the reagent can be readily regenerated.

Isoindolinone inhibitors of the murine double minute 2 (MDM2)-p53 protein-protein interaction: structure-activity studies leading to improved potency.

Inhibition of the MDM2-p53 interaction has been shown to produce an antitumor effect, especially in MDM2 amplified tumors. The isoindolinone scaffold has proved to be versatile for the discovery of MDM2-p53 antagonists. Optimization of previously reported inhibitors, for example, NU8231 (7) and NU8165 (49), was guided by MDM2 NMR titrations, which indicated key areas of the binding interaction to be explored. Variation of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification of 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhibitor (IC(50) = 0.23 ± 0.01 µM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (+)-R-enantiomer (74a; IC(50) = 0.17 ± 0.02 µM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for wild-type p53 cell lines in growth inhibition assays.