Exploration of macrocyclic peptide binders to the extracellular CRD domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

Elevated levels of receptor tyrosine kinase-like orphan receptor 1 (RORl) expression are observed in multiple hematological and solid tumors, but not in most of the healthy adult tissues, identifying ROR1 as an attractive target for tumor-specific therapy. Herein we will describe the discovery of macrocyclic peptides as binders of the extracellular Cysteine-Rich Domain (CRD) of human ROR1 via mRNA in vitro selection technology using the PDPS platform, followed by exploration of sidechain SAR of parent macrocycle peptides, fluorescently labeled analogs, and a Peptide Drug Conjugate (PDC). The parent macrocyclic peptides represented by Compound 1 and Compound 14 displayed nanomolar cell-based binding to ROR1 and relatively good internalization in 786-O and MDA-MB-231 tumor cell lines. However, these peptides were not observed to induce apoptosis in Mia PaCa-2 cells, a model pancreatic tumor cell line with a relatively low level of cell surface expression of ROR1.

Difluorocyclobutylacetylenes as positive allosteric modulators of mGluR5 with reduced bioactivation potential.

Schizophrenia is a serious illness that affects millions of patients and has been associated with N-methyl-d-aspartate receptor (NMDAR) hypofunction. It has been demonstrated that activation of metabotropic glutamate receptor 5 (mGluR5) enhances NMDA receptor function, suggesting the potential utility of mGluR5 positive allosteric modulators (PAMs) in the treatment of schizophrenia. Herein we describe the optimization of an mGluR5 PAM by replacement of a phenyl with aliphatic heterocycles and carbocycles as a strategy to reduce bioactivation in a biaryl acetylene chemotype. Replacement with a difluorocyclobutane followed by further optimization culminated in the identification of compound 32, a low fold shift PAM with reduced bioactivation potential. Compound 32 demonstrated favorable brain uptake and robust efficacy in mouse novel object recognition (NOR) at low doses.

Late-stage optimization of a tercyclic class of S1P3-sparing, S1P1 receptor agonists.

Poor solubility and cationic amphiphilic drug-likeness were liabilities identified for a lead series of S1P3-sparing, S1P1 agonists originally developed from a high-throughput screening campaign. This work describes the subsequent optimization of these leads by balancing potency, selectivity, solubility and overall molecular charge. Focused SAR studies revealed favorable structural modifications that, when combined, produced compounds with overall balanced profiles. The low brain exposure observed in rat suggests that these compounds would be best suited for the potential treatment of peripheral autoimmune disorders.

Discovery of 1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxide analogs as new RORC modulators.

Structure-based and pharmacophore-based virtual screening in combination with combinatorial chemistry and X-ray crystallography led to the discovery of a new class of benzothiadiazole dioxide analogs with functional activity as RORC inverse agonists. The early RORC SAR compound 14 exhibited RORC inhibition in a cell based reporter gene assay of 5.7 µM and bound to RORC with an affinity of 1.6 µM in a fluorescence polarization assay displacing a ligand binding site probe. Crystallography confirmed the binding mode of the compound in the ligand binding domain displaying the engagement of a novel sub pocket close to Ser404. Subsequent optimization yielded compounds with enhanced RORC inverse agonist activity. The most active compound 19 showed an IC50 of 440 nM in a human PBMC assay.

Piperazinyl-oxadiazoles as selective sphingosine-1-phosphate receptor agonists.

The discovery of a new series of selective S1P1 agonists is described. This series of piperazinyl-oxadiazole derivatives was rapidly optimized starting from high-throughput screening hit 1 to afford potent and selective lead compound 10d. Further SAR studies showed that 10d was converted to the active phosphate metabolite 29 in vivo. Oral administration of compound 10d to rats was shown to induce lymphopenia at 3 mg/kg.

Benzimidazolone as potent chymase inhibitor: modulation of reactive metabolite formation in the hydrophobic (P1) region.

A new class of chymase inhibitor featuring a benzimidazolone core with an acid side chain and a P(1) hydrophobic moiety is described. Incubation of the lead compound with GSH resulted in the formation of a GSH conjugate on the benzothiophene P(1) moiety. Replacement of the benzothiophene with different heterocyclic systems such as indoles and benzoisothiazole is feasible. Among the P(1) replacements, benzoisothiazole prevents the formation of GSH conjugate and an in silico analysis of oxidative potentials agreed with the experimental outcome.

Indole Inhibitors of MMP-13 for Arthritic Disorders.

Here, we described the design, by fragment merging and multiparameter optimization, of selective MMP-13 inhibitors that display an appropriate balance of potency and physicochemical properties to qualify as tool compounds suitable for in vivo testing. Optimization of potency was guided by structure-based insights, specifically to replace an ester moiety and introduce polar directional hydrogen bonding interactions in the core of the molecule. By introducing polar enthalpic interactions in this series of inhibitors, the overall beneficial physicochemical properties were maintained. These physicochemical properties translated to excellent drug-like properties beyond potency. In a murine model of rheumatoid arthritis, treatment of mice with selective inhibitors of MMP-13 resulted in a statistically significant reduction in the mean arthritic score vs control when dosed over a 14 day period.

SAR studies of non-zinc-chelating MMP-13 inhibitors: improving selectivity and metabolic stability.

SAR studies to improve the selectivity and metabolic stability of a class of recently discovered MMP-13 inhibitors are reported. Improved selectivity was achieved by modifying interactions with the S1' pocket. Metabolic stability was improved through reduction of inhibitor lipophilicity. This translated into lower in vivo clearance for the preferred compound.

Improving potency and selectivity of a new class of non-Zn-chelating MMP-13 inhibitors.

Discovery and optimization of potency and selectivity of a non-Zn-chelating MMP-13 inhibitor with the aid of protein co-crystal structural information is reported. This inhibitor was observed to have a binding mode distinct from previously published MMP-13 inhibitors. Potency and selectivity were improved by extending the hit structure out from the active site into the S1' pocket.

Imidazo[1,2-a]pyrazine diaryl ureas: inhibitors of the receptor tyrosine kinase EphB4.

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent and Nexavar, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.

Development of 1H-Pyrazolo[3,4-b]pyridines as Metabotropic Glutamate Receptor 5 Positive Allosteric Modulators.

The metabotropic glutamate receptor 5 (mGluR5) is an attractive target for the treatment of schizophrenia due to its role in regulating glutamatergic signaling in association with the N-methyl-d-aspartate receptor (NMDAR). We describe the synthesis of 1H-pyrazolo[3,4-b]pyridines and their utility as mGluR5 positive allosteric modulators (PAMs) without inherent agonist activity. A facile and convergent synthetic route provided access to a structurally diverse set of analogues that contain neither the aryl-acetylene-aryl nor aryl-methyleneoxy-aryl elements, the predominant structural motifs described in the literature. Binding studies suggest that members of our new chemotype do not engage the receptor at the MPEP and CPPHA mGluR5 allosteric sites. SAR studies culminated in the first non-MPEP site PAM, 1H-pyrazolo[3,4-b]pyridine 31 (BMT-145027), to improve cognition in a preclinical rodent model of learning and memory.

No differences observed among multiple clinical S1P1 receptor agonists (functional antagonists) in S1P1 receptor down-regulation and degradation.

Sphingosine-1-phosphate (S1P) is a bioactive metabolite with pleiotropic effects on multiple cellular processes in health and disease. Responses elicited by S1P are a result of binding to five specific G-protein-coupled receptors. We have developed multiple assays to systematically study the downstream signaling of these receptors, including early events such as direct receptor activation (GTPγS) as well as more distal events such as S1P1 receptor degradation. Employing such assays, we have characterized and compared multiple S1P1 agonists that are in clinical development including FTY720, BAF312, CS-0777, and other molecules from the S1P1 patent literature. Our parallel assessment has allowed us to compare their potency against S1P1, their selectivity against the four other S1P receptors, as well as species cross-reactivity. We note that all of the compounds studied signal in an identical manner through S1P1, leading to receptor degradation.

Discovery of potent, selective chymase inhibitors via fragment linking strategies.

Chymase plays an important and diverse role in the homeostasis of a number of cardiovascular processes. Herein, we describe the identification of potent, selective chymase inhibitors, developed using fragment-based, structure-guided linking and optimization techniques. High-concentration biophysical screening methods followed by high-throughput crystallography identified an oxindole fragment bound to the S1 pocket of the protein exhibiting a novel interaction pattern hitherto not observed in chymase inhibitors. X-ray crystallographic structures were used to guide the elaboration/linking of the fragment, ultimately leading to a potent inhibitor that was >100-fold selective over cathepsin G and that mitigated a number of liabilities associated with poor physicochemical properties of the series it was derived from.

Bioluminescent method for assaying multiple semicarbazide-sensitive amine oxidase (SSAO) family members in both 96- and 384-well formats.

Vascular adhesion protein-1 (VAP-1), also known as semicarbazide-sensitive amine oxidase (SSAO) or copper-containing amine oxidase (AOC3, EC 1.4.3.6), catalyzes oxidative deamination of primary amines. One endogenous substrate has recently been described (Siglec 10), and although its mechanism of action in vivo is not completely understood, it is suggested to play a role in immune cell trafficking, making it a target of interest for autoimmune and inflammatory diseases. Much of the enzymology performed around this target has been conducted with absorbance, fluorescent, or radiometric formats that can have some limitations for high-throughput screening and subsequent compound profiling. The authors present the use of a bioluminescent assay, originally developed for monoamine oxidase enzymes, in a high-throughput format. It can be used for related SSAOs such as AOC1 given their substrate similarity with VAP-1. The authors also demonstrate that it is compatible with different sources of VAP-1, both purified recombinant and VAP-1 overexpressed on live cells.

Fragment-based discovery of indole inhibitors of matrix metalloproteinase-13.

Matrix metalloproteases (MMPs) play an important role in cartilage homeostasis under both normal and inflamed disease states and, thus, have become attractive targets for the treatment of arthritic diseases. Herein, we describe the identification of a potent, selective MMP-13 inhibitor, developed using fragment-based structure-guided lead identification and optimization techniques. Virtual screening methods identified a novel, indole-based MMP-13 inhibitor that bound into the S1' pocket of the protein exhibiting a novel interaction pattern hitherto not observed in MMP-13 inhibitors. X-ray crystallographic structures were used to guide the elaboration of the fragment, ultimately leading to a potent inhibitor that was >100-fold selective over nine other MMP isoforms tested.