Incorporation of Intracellular NanoSIMS Tracers to Oligonucleotide Conjugates via Strain Promoted Sydnone-Alkyne Cycloaddition.

Extensive efforts have been dedicated to developing cell-specific targeting ligands that can be conjugated to therapeutic cargo, offering a promising yet still challenging strategy to deliver oligonucleotide therapeutics beyond the liver. Indeed, while the cargo and the ligand are crucial, the third component, the linker, is integral but is often overlooked. Here, we present strain-promoted sydnone-alkyne cycloaddition as a versatile linker chemistry for oligonucleotide synthesis, expanding the choices for bioconjugation of therapeutics while enabling subcellular detection of the linker and payload using nanoscale secondary ion mass spectrometry (NanoSIMS) imaging. This strategy was successfully applied to peptide and lipid ligands and profiled using the well characterized N-acetylgalactosamine (GalNAc) targeting ligand. The linker did not affect the expected activity of the conjugate and was detectable and distinguishable from the labeled cargo. Finally, this work not only offers a practical bioconjugation method but also enables the assessment of the linker's subcellular behavior, facilitating NanoSIMS imaging to monitor the three key components of therapeutic conjugates.

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.

Synthesis of Diverse α-Fluoroalkoxyaryl Derivatives and Their Use for the Generation of Fluorinated Macrocycles.

Introduction of difluorinated functionality has emerged as a powerful means for conformational design with minimal steric footprint. Synthetic approaches for the preparation of aryl difluoromethylene ether containing novel building blocks were established, enabling the inclusion of the aryl difluoromethylene ether system into macrocyclic scaffolds for the first time.

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.

Fluorine-Directed Glycosylation Enables the Stereocontrolled Synthesis of Selective SGLT2 Inhibitors for Type†II Diabetes.

Inhibition of the sodium-glucose co-transporters (SGLT1 and SGLT2) is a validated strategy to address the increasing prevalence of type II diabetes mellitus. However, achieving selective inhibition of human SGLT1 or SGLT2 remains challenging. Orally available small molecule drugs based on the d-glucose core of the natural product Gliflozin have proven to be clinically effective in this regard, effectively impeding glucose reabsorption. Herein, we disclose the influence of molecular editing with fluorine at the C2 position of the pyranose ring of Phlorizin analogues Remogliflozin Etabonate and Dapagliflozin (Farxiga® ) to concurrently direct ß-selective glycosylation, as is required for biological efficacy, and enhance aspects of the physicochemical profile. Given the abundance of glycosylated pharmaceuticals in diabetes therapy that contain a ß-configured d-glucose nucleus, it is envisaged that this strategy may prove to be expansive.

Design, synthesis, and SAR of a series of activated protein C (APC) inhibitors with selectivity against thrombin for the treatment of haemophilia.

A design strategy was used to identify inhibitors of activated protein C with selectivity over thrombin featured by a basic and/or aromatic functionality for binding to the S2 pocket. Our strongest inhibitor showed an IC50-material value and selectivity for APC vs thrombin similar to a compound previously reported in the literature. However, in contrast to the reference compound, our compound showed a retained coagulant effect of thrombin with increasing substrate concentration in a modified Calibrated Automated Thrombogram (CAT) method. This was likely related to our compound being inactive against FVIIa, while the reference compound showed an IC50 of 8.9 µM. Thus, the higher selectivity of our compound against all relevant coagulation factors likely explained its higher therapeutic potential in comparison to the reference compound. The data indicate that at least a 100-fold selectivity over other serine proteases in the coagulation cascade will be required for an effective APC inhibitor.

Discovery of a novel pyrazole series of group X secreted phospholipase A2 inhibitor (sPLA2X) via fragment based virtual screening.

The discovery of potent novel pyrazole containing group X secreted phospholipase A2 inhibitors via structure based virtual screening is reported. Docking was applied on a large set of in-house fragment collection and pharmacophore feature matching was used to filter docking poses. The selected virtual screening hits was run in NMR screening, a potent pyrazole containing fragment hit was identified and confirmed by its complex X-ray structure and the following biochemical assay result. Expansion on the fragment hit has led to further improvement of potency while maintaining high ligand efficiency, thus supporting the further development of this chemical series.

Heart regeneration: opportunities and challenges for drug discovery with novel chemical and therapeutic methods or agents.

Following a heart attack, more than a billion cardiac muscle cells (cardiomyocytes) can be killed, leading to heart failure and sudden death. Much research in this area is now focused on the regeneration of heart tissue through differentiation of stem cells, proliferation of existing cardiomyocytes and cardiac progenitor cells, and reprogramming of fibroblasts into cardiomyocytes. Different chemical modalities (i.e. methods or agents), ranging from small molecules and RNA approaches (including both microRNA and anti-microRNA) to modified peptides and proteins, are showing potential to meet this medical need. In this Review, we outline the recent advances in these areas and describe both the modality and progress, including novel screening strategies to identify hits, and the upcoming challenges and opportunities to develop these hits into pharmaceuticals, at which chemistry plays a key role.

Discovery of N-[[1-[2-(tert-butylcarbamoylamino)ethyl]-4-(hydroxymethyl)-4-piperidyl]methyl]-3,5-dichloro-benzamide as a selective T-type calcium channel (Cav3.2) inhibitor.

The T-type calcium channel inhibitor Mibefradil was reported to protect the heart from atrial remodeling, a key process involved in the development of atrial fibrillation and arrhythmias. Mibefradil is not a selective T-type calcium channel inhibitor and also affects the function of different ion channels. Our aim was to develop a selective T-type calcium channel inhibitor to validate the importance of T-type-related pharmacology in atrial fibrillation. Structural optimisation of a previously disclosed hit series focussed on minimising exposure to the central nervous system and improving pharmacokinetic properties, while maintain adequate potency and selectivity. This resulted in the design of N-[[1-[2-(tert-butylcarbamoylamino)ethyl]-4-(hydroxymethyl)-4-piperidyl]methyl]-3,5-dichloro-benzamide, a novel, selective, peripherally restricted chemical probe to verify the role of T-type calcium channel inhibition on atrial fibrillation protection.

A structure-specific small molecule inhibits a miRNA-200 family member precursor and reverses a type 2 diabetes phenotype.

MicroRNA families are ubiquitous in the human transcriptome, yet targeting of individual members is challenging because of sequence homology. Many secondary structures of the precursors to these miRNAs (pri- and pre-miRNAs), however, are quite different. Here, we demonstrate both in vitro and in cellulis that design of structure-specific small molecules can inhibit a particular miRNA family member to modulate a disease pathway. The miR-200 family consists of five miRNAs, miR-200a, -200b, -200c, -141, and -429, and is associated with type 2 diabetes (T2D). We designed a small molecule that potently and selectively targets pre-miR-200c's structure and reverses a pro-apoptotic effect in a pancreatic ß cell model. In contrast, an oligonucleotide targeting the RNA's sequence inhibited all family members. Global proteomics and RNA sequencing analyses further demonstrate selectivity for miR-200c. Collectively, these studies establish that miR-200c plays an important role in T2D, and small molecules targeting RNA structure can be an important complement to oligonucleotides.

Late-Stage Functionalization with Cysteine Staples Generates Potent and Selective Melanocortin Receptor-1 Agonists.

In this work, cysteine staples were used as a late-stage functionalization strategy to diversify peptides and build conjugates targeting the melanocortin G-protein-coupled receptors [melanocortin receptor-1 (MC1R) and MC3R-MC5R]. Monocyclic and bicyclic agonists based on sunflower trypsin inhibitor-1 were used to generate a selection of stapled peptides that were evaluated for binding (pKi) and functional activation (pEC50) of the melanocortin receptor subtypes. Stapled peptides generally had improved activity, with aromatic stapled peptides yielding selective MC1R agonists, including a xylene-stapled peptide (2) with an EC50 of 1.9 nM for MC1R and >150-fold selectivity for MC3R and MC4R. Selected stapled peptides were further functionalized with linkers and payloads, generating a series of conjugated peptides with potent MC1R activity, including one pyridazine-functionalized peptide (21) with picomolar activity at MC1R (Ki 58 pM; EC50 < 9 pM). This work demonstrates that staples can be used as modular synthetic tools to tune potency and selectivity in peptide-based drug design.

Discovery of N-(1-adamantyl)-2-(4-alkylpiperazin-1-yl)acetamide derivatives as T-type calcium channel (Cav3.2) inhibitors.

Chemical evolution of a HTS-based fragment hit resulted in the identification of N-(1-adamantyl)-2-[4-(2-tetrahydropyran-4-ylethyl)piperazin-1-yl]acetamide, a novel, selective T-type calcium channel (Ca(v)3.2) inhibitor with in vivo antihypertensive effect in rats.

Melanocortin 1 Receptor Agonists Based on a Bivalent, Bicyclic Peptide Framework.

We have designed a new class of highly potent bivalent melanocortin receptor ligands based on the nature-derived bicyclic peptide sunflower trypsin inhibitor 1 (SFTI-1). Incorporation of melanotropin pharmacophores in each of the two turn regions of SFTI-1 resulted in substantial gains in agonist activity particularly at human melanocortin receptors 1 and 3 (hMC1R/hMC3R) compared to monovalent analogues. In in vitro binding and functional assays, the most potent molecule, compound 6, displayed low picomolar agonist activity at hMC1R (pEC50 > 10.3; EC50 < 50 pM; pKi: 10.16 ± 0.04; Ki: 69 ± 5 pM) and is at least 30-fold more selective for this receptor than for hMC3R, hMC4R, or hMC5R. The results are discussed in the context of structural homology models of hMCRs in complex with the developed bivalent ligands.

Target-Directed Approaches for Screening Small Molecules against RNA Targets.

RNA molecules have a variety of cellular functions that can drive disease pathologies. They are without a doubt one of the most intriguing yet controversial small-molecule drug targets. The ability to widely target RNA with small molecules could be revolutionary, once the right tools, assays, and targets are selected, thereby defining which biomolecules are targetable and what constitutes drug-like small molecules. Indeed, approaches developed over the past 5-10 years have changed the face of small molecule-RNA targeting by addressing historic concerns regarding affinity, selectivity, and structural dynamics. Presently, selective RNA-protein complex stabilizing drugs such as branaplam and risdiplam are in clinical trials for the modulation of SMN2 splicing, compounds identified from phenotypic screens with serendipitous outcomes. Fully developing RNA as a druggable target will require a target engagement-driven approach, and evolving chemical collections will be important for the industrial development of this class of target. In this review we discuss target-directed approaches that can be used to identify RNA-binding compounds and the chemical knowledge we have today of small-molecule RNA binders.

Design of a small molecule that stimulates vascular endothelial growth factor A enabled by screening RNA fold-small molecule interactions.

Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis in human endothelial cells, and increasing its expression is a potential treatment for heart failure. Here, we report the design of a small molecule (TGP-377) that specifically and potently enhances VEGFA expression by the targeting of a non-coding microRNA that regulates its expression. A selection-based screen, named two-dimensional combinatorial screening, revealed preferences in small-molecule chemotypes that bind RNA and preferences in the RNA motifs that bind small molecules. The screening program increased the dataset of known RNA motif-small molecule binding partners by 20-fold. Analysis of this dataset against the RNA-mediated pathways that regulate VEGFA defined that the microRNA-377 precursor, which represses Vegfa messenger RNA translation, is druggable in a selective manner. We designed TGP-377 to potently and specifically upregulate VEGFA in human umbilical vein endothelial cells. These studies illustrate the power of two-dimensional combinatorial screening to define molecular recognition events between 'undruggable' biomolecules and small molecules, and the ability of sequence-based design to deliver efficacious structure-specific compounds.

IA-Lab: A MATLAB framework for efficient microscopy image analysis development, applied to quantifying intracellular transport of internalized peptide-drug conjugate.

This work presents a MATLAB-based software package for high-throughput microscopy image analysis development, making such development more accessible for a large user community. The toolbox provides a GUI and a number of analysis workflows, and can serve as a general framework designed to allow for easy extension. For a new application, only a minor part of the object-oriented code needs to be replaced by new components, making development efficient. This makes it possible to quickly develop solutions for analysis not available in existing tools. We show its use in making a tool for quantifying intracellular transport of internalized peptide-drug conjugates. The code is freely available as open source on GitHub (https://github.com/amcorrigan/ia-lab).

Stereocontrolled Synthesis of Tetrafluoropentanols: Multivicinal Fluorinated Alkane Units for Drug Discovery.

A stereodivergent synthesis of four diastereomeric 2,3,4,5-tetrafluoropentanols is disclosed. X-ray crystallographic analysis reveals conformations that manifest sequential stereoelectronic gauche effects (σC-H/C → σC-F*), thereby generating topological diversity via subtle C(sp3)-H to C(sp3)-F exchange. Two representative tetrafluoro arrays have been incorporated into truncated analogues of Gilenya for the management of relapsing remitting multiple sclerosis. These closely similar multivicinal fluoroalkanes have notably different physicochemical profiles and were found to be stable in the presence of human microsomes.

Imidazopyridines as VLA-4 integrin antagonists.

We describe a novel series of imidazopyridine substituted phenylalanines which are potent VLA-4 antagonists. A wide variety of substituents are tolerated as replacements for the pendant 3-pyridyl ring. A clear structure-activity relationship was identified around the substitution of the 3-amino-cyclobut-2-enone portion of the molecule.

Exploring physicochemical space via a bioisostere of the trifluoromethyl and ethyl groups (BITE): attenuating lipophilicity in fluorinated analogues of Gilenya® for multiple sclerosis.

The direct, catalytic vicinal difluorination of terminal alkenes via an I(i)/I(iii) manifold was exploited to install a chiral, hybrid bioisostere of the CF3 and Et groups (BITE) in Gilenya®; the first orally available drug for the clinical management of Multiple Sclerosis (MS). This subtle fluorination pattern allows lipophilicity (log D) to be tempered compared to the corresponding CF3 and Et derivatives (CH2CH3 > CH2CF3 > CHFCH2F).

The Conundrum of GSK3 Inhibitors: Is it the Dawn of a New Beginning?

Spanning over three decades of extensive drug discovery research, the efforts to develop a potent and selective GSK3 inhibitor as a therapeutic for the treatment of type 2 diabetes, Alzheimer's disease (AD), bipolar disorders and cancer have been futile. Since its initial discovery in 1980 and subsequent decades of research, one cannot underscore the importance of the target and the promise of a game changing disease modifier. Several pharmaceutical companies, biotech companies, and academic institutions raged in a quest to unravel the biology and discover potent and selective GSK3 inhibitors, some of which went through clinical trials. However, the conundrum of what happened to the fate of the AstraZeneca's GSK3 inhibitors and the undertaking to find a therapeutic that could control glycogen metabolism and aberrant tau hyperphosphorylation in the brain, and rescue synaptic dysfunction has largely been untold. AstraZeneca was in the forefront of GSK3 drug discovery research with six GSK3 drug candidates, one of which progressed up to Phase II clinical trials in the quest to untangle the tau hypothesis for AD. Analysis of key toxicity issues, serendipitous findings and efficacy, and biomarker considerations in relation to safety margins have limited the potential of small molecule therapeutics as a way forward. To guide future innovation of this important target, we reveal the roller coaster journey comprising of two decades of preclinical and clinical GSK3 drug discovery at AstraZeneca; the understanding of which could lead to improved GSK3 therapies for disease. These learnings in combination with advances in achieving kinase selectivity, different modes of action as well as the recent discovery of novel conjugated peptide technology targeting specific tissues have potentially provided a venue for scientific innovation and a new beginning for GSK3 drug discovery.