Development of Potent Mcl-1 Inhibitors: Structural Investigations on Macrocycles Originating from a DNA-Encoded Chemical Library Screen.

Evasion of apoptosis is critical for the development and growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family, associated with tumor aggressiveness, poor survival, and drug resistance. Development of Mcl-1 inhibitors implies blocking of protein-protein interactions, generally requiring a lengthy optimization process of large, complex molecules. Herein, we describe the use of DNA-encoded chemical library synthesis and screening to directly generate complex, yet conformationally privileged macrocyclic hits that serve as Mcl-1 inhibitors. By applying a conceptual combination of conformational analysis and structure-based design in combination with a robust synthetic platform allowing rapid analoging, we optimized in vitro potency of a lead series into the low nanomolar regime. Additionally, we demonstrate fine-tuning of the physicochemical properties of the macrocyclic compounds, resulting in the identification of lead candidates 57/59 with a balanced profile, which are suitable for future development toward therapeutic use.

Novel irreversible covalent BTK inhibitors discovered using DNA-encoded chemistry.

Libraries of DNA-Encoded small molecules created using combinatorial chemistry and synthetic oligonucleotides are being applied to drug discovery projects across the pharmaceutical industry. The majority of reported projects describe the discovery of reversible, i.e. non-covalent, target modulators. We synthesized multiple DNA-encoded chemical libraries terminated in electrophiles and then used them to discover covalent irreversible inhibitors and report the successful discovery of acrylamide- and epoxide-terminated Bruton's Tyrosine Kinase (BTK) inhibitors. We also demonstrate their selectivity, potency and covalent cysteine engagement using a range of techniques including X-ray crystallography, thermal transition shift assay, reporter displacement assay and intact protein complex mass spectrometry. The epoxide BTK inhibitors described here are the first ever reported to utilize this electrophile for this target.

Discovery of 2,4-1H-Imidazole Carboxamides as Potent and Selective TAK1 Inhibitors.

Herein we report the discovery of 2,4-1H-imidazole carboxamides as novel, biochemically potent, and kinome selective inhibitors of transforming growth factor ß-activated kinase 1 (TAK1). The target was subjected to a DNA-encoded chemical library (DECL) screen. After hit analysis a cluster of compounds was identified, which was based on a central pyrrole-2,4-1H-dicarboxamide scaffold, showing remarkable kinome selectivity. A scaffold-hop to the corresponding imidazole resulted in increased biochemical potency. Next, X-ray crystallography revealed a distinct binding mode compared to other TAK1 inhibitors. A benzylamide was found in a perpendicular orientation with respect to the core hinge-binding imidazole. Additionally, an unusual amide flip was observed in the kinase hinge region. Using structure-based drug design (SBDD), key substitutions at the pyrrolidine amide and the glycine resulted in a significant increase in biochemical potency.

Full-length in meso structure and mechanism of rat kynurenine 3-monooxygenase inhibition.

The structural mechanisms of single-pass transmembrane enzymes remain elusive. Kynurenine 3-monooxygenase (KMO) is a mitochondrial protein involved in the eukaryotic tryptophan catabolic pathway and is linked to various diseases. Here, we report the mammalian full-length structure of KMO in its membrane-embedded form, complexed with compound 3 (identified internally) and compound 4 (identified via DNA-encoded chemical library screening) at 3.0 Å resolution. Despite predictions suggesting that KMO has two transmembrane domains, we show that KMO is actually a single-pass transmembrane protein, with the other transmembrane domain lying laterally along the membrane, where it forms part of the ligand-binding pocket. Further exploration of compound 3 led to identification of the brain-penetrant compound, 5. We show that KMO is dimeric, and that mutations at the dimeric interface abolish its activity. These results will provide insight for the drug discovery of additional blood-brain-barrier molecules, and help illuminate the complex biology behind single-pass transmembrane enzymes.

Novel Autotaxin Inhibitor for the Treatment of Idiopathic Pulmonary Fibrosis: A Clinical Candidate Discovered Using DNA-Encoded Chemistry.

The activity of the secreted phosphodiesterase autotaxin produces the inflammatory signaling molecule LPA and has been associated with a number of human diseases including idiopathic pulmonary fibrosis (IPF). We screened a single DNA-encoded chemical library (DECL) of 225 million compounds and identified a series of potent inhibitors. Optimization of this series led to the discovery of compound 1 (X-165), a highly potent, selective, and bioavailable small molecule. Cocrystallization of compound 1 with human autotaxin demonstrated that it has a novel binding mode occupying both the hydrophobic pocket and a channel near the autotaxin active site. Compound 1 inhibited the production of LPA in human and mouse plasma at nanomolar levels and showed efficacy in a mouse model of human lung fibrosis. After successfully completing IND-enabling studies, compound 1 was approved by the FDA for a Phase I clinical trial. These results demonstrate that DECL hits can be readily optimized into clinical candidates.

Discovery of a Potent BTK Inhibitor with a Novel Binding Mode by Using Parallel Selections with a DNA-Encoded Chemical Library.

We have identified and characterized novel potent inhibitors of Bruton's tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets.

Improving cellular adhesion on scaffolds for transplantation: synthesising a poly(MMA-co-PEGM) network.

Electrospun fibrous matrices prepared from methacrylate-based copolymers are investigated as a tool for retinal pigment epithelium (RPE) transplantation in the treatment of degenerative retinal diseases. Human RPE cells were used to probe the cell-surface interactions on these copolymer matrices. For the first time, simple changes in chemical functionality have been found to induce gel formation of these methacrylate backbone copolymers in vitro. This effect is shown to significantly improve RPE cell adhesion and survival.

Developing methacrylate-based copolymers as an artificial Bruch's membrane substitute.

Age-related macular degeneration (AMD) is the most common cause of blindness in the developed world. There is currently no treatment for the cellular loss, which is characteristic of AMD. Transplantation of retinal pigment epithelium (RPE) cells represents a potential therapy. Because of AMD-related pathology in the native support, Bruch's membrane, transplanted RPE cells require a scaffold to reside on. We present here the development of an electrospun fibrous scaffold derived from methyl methacrylate and poly(ethylene glycol) (PEG) methacrylate for novel application as an RPE scaffold. Scaffolds were chemically modified to improve cell adhesion by functionalization not previously reported for this type of copolymer system. A human RPE cell line was used to investigate cell-scaffold interactions for up to two weeks in vitro. Scanning electron microscopy was used to characterize the fibrous scaffolds and confirm cell attachment. By day 15, cell area was significantly (p < 0.001) enhanced on scaffolds with chemical modification of the PEG chain terminus. In addition, significantly, less-apoptotic cell death was demonstrable on these modified surfaces.

The chemistry of retinal transplantation: the influence of polymer scaffold properties on retinal cell adhesion and control.

Age-related macular degeneration is the most common cause of blindness in the UK. Cellular replacement of retinal pigment epithelium cells is a potential therapeutic option to treat the cellular loss and dysfunction which is characteristic of age-related macular degeneration and other progressive retinopathies. A supportive scaffold, natural or artificial, may be required to facilitate cell delivery to the eye. Research to improve the biomimetic properties of such scaffolds, in order to optimise cell attachment and functionality following implantation, is ongoing. This short review will focus on the potential of biomaterials for ocular tissue engineering and how surface modification and the physical properties of these scaffolds can be tailored to help realise the full clinical potential of retinal pigment epithelium cell transplantation.

Optimisation of polymer scaffolds for retinal pigment epithelium (RPE) cell transplantation.

AIM: To evaluate a variety of copolymers as suitable scaffolds to facilitate retinal pigment epithelium (RPE) transplantation. METHODS: Five blends of poly(l-lactic acid) (PLLA) with poly(d,l-lactic-glycolic acid) (PLGA) were manufactured by a solid-liquid phase separation technique. The blends were 10:90, 25:75, 50:50, 75:25 and 90:10 (PLLA:PLGA). All blend ratios were validated by nuclear magnetic resonance spectroscopy. Samples of polymer blends were coated with laminin. Coated and uncoated blends were seeded with a human RPE cell line. Cell attachment, viability and retention of phenotype were assessed. RESULTS: As the lactide unit content increased pore size generally became smaller. The 25:75 PLLA:PLGA blend was the most porous (44%) and thinnest (134 µm) scaffold produced. ARPE-19 cells retained an appropriate phenotype with minimal cell death for up to 4 weeks in vitro. Cell density was maintained on only one of the fabricated ratios (25% PLLA:75% PLGA). A consistent decrease in apoptotic cell death with time was observed on coated samples of this blend. A decrease in polymer thickness concomitant with an increase in porosity characteristic of degradation was observed with all polymer blends. CONCLUSIONS: This study demonstrates that a 25:75 copolymer blend of PLLA:PLGA is a potentially useful scaffold for ocular cell transplantation.