Probing the Binding Mechanism of Acylated Peptides to Human Serum Albumin.

Peptides represent an increasingly important class of pharmaceutical products. During the last decade or so, acylation with fatty acids has demonstrated considerable success in prolonging the circulating half-life of therapeutic peptides by exploiting the ability of fatty acids to reversibly bind to human serum albumin (HSA), thus significantly impacting their pharmacological profiles. Employing methyl-13C-labeled oleic acid or palmitic acid as probe molecules and exploiting HSA mutants designed to probe fatty acid binding, the signals in two-dimensional (2D) nuclear magnetic resonance (NMR) spectra corresponding to high-affinity fatty acid binding sites in HSA were assigned. Subsequently, using a set of selected acylated peptides, competitive displacement experiments by 2D NMR identified a primary fatty acid binding site in HSA utilized in acylated peptide binding. These results represent an important first step toward understanding the structural basis for acylated peptides binding to HSA.

Defining Target Engagement Required for Efficacy In Vivo at the Retinoic Acid Receptor-Related Orphan Receptor C2 (RORγt).

The Th17 pathway has been implicated in autoimmune diseases. The retinoic acid receptor-related orphan receptor C2 (RORγt) is a master regulator of Th17 cells and controls the expression of IL-17A. RORγt is expressed primarily in IL-17A-producing lymphoid cells. Here we describe a virtual screen of the ligand-binding pocket and subsequent screen in a binding assay that identified the 1-benzyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-2'-carboxamide scaffold as a starting point for optimization of binding affinity and functional activity guided by structure-based design. Compound 12 demonstrated activity in a mouse PK/PD model and efficacy in an inflammatory arthritis mouse model that were used to define the level and duration of target engagement required for efficacy in vivo. Further optimization to improve ADME and physicochemical properties with guidance from simulations and modeling provided compound 22, which is projected to achieve the level and duration of target engagement required for efficacy in the clinic.

Discovery and characterization of a neutralizing pan-ELR+CXC chemokine monoclonal antibody.

CXCR1 and CXCR2 signaling play a critical role in neutrophil migration, angiogenesis, and tumorigenesis and are therefore an attractive signaling axis to target in a variety of indications. In human, a total of seven chemokines signal through these receptors and comprise the ELR+CXC chemokine family, so named because of the conserved ELRCXC N-terminal motif. To fully antagonize CXCR1 and CXCR2 signaling, an effective therapeutic should block either both receptors or all seven ligands, yet neither approach has been fully realized clinically. In this work, we describe the generation and characterization of LY3041658, a humanized monoclonal antibody that binds and neutralizes all seven human and cynomolgus monkey ELR+CXC chemokines and three of five mouse and rat ELR+CXC chemokines with high affinity. LY3041658 is able to block ELR+CXC chemokine-induced Ca2+ mobilization, CXCR2 internalization, and chemotaxis in vitro as well as neutrophil mobilization in vivo without affecting other neutrophil functions. In addition to the in vitro and in vivo activity, we characterized the epitope and structural basis for binding in detail through alanine scanning, crystallography, and mutagenesis. Together, these data provide a robust preclinical characterization of LY3041658 for which the efficacy and safety is being evaluated in human clinical trials for neutrophilic skin diseases.

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage.

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.

N+1 Engineering of an Aspartate Isomerization Hotspot in the Complementarity-Determining Region of a Monoclonal Antibody.

Aspartate (Asp) isomerization is a common degradation pathway and a potential critical quality attribute that needs to be well characterized during the optimization and development of therapeutic antibodies. A putative Asp-serine (Ser) isomerization motif was identified in the complementarity-determining region of a humanized monoclonal antibody and shown to be a developability risk using accelerated stability analyses. To address this issue, we explored different antibody engineering strategies. Direct engineering of the Asp residue resulted in a greater than 5× loss of antigen-binding affinity and bioactivity, indicating a critical role for this residue. In contrast, rational engineering of the Ser residue at the n+1 position had a negligible impact on antigen binding affinity and bioactivity compared with the parent molecule. Furthermore, the n+1 engineering strategy effectively eliminated Asp isomerization as determined by accelerated stability analysis. This outcome affirms that the rate of Asp isomerization is strongly dependent on the identity of the n+1 residue. This report highlights a systematic antibody engineering strategy for mitigating an Asp isomerization developability risk during lead optimization.

Novel Autotaxin Inhibitors for the Treatment of Osteoarthritis Pain: Lead Optimization via Structure-Based Drug Design.

In an effort to develop a novel therapeutic agent aimed at addressing the unmet need of patients with osteoarthritis pain, we set out to develop an inhibitor for autotaxin with excellent potency and physical properties to allow for the clinical investigation of autotaxin-induced nociceptive and neuropathic pain. An initial hit identification campaign led to an aminopyrimidine series with an autotaxin IC50 of 500 nM. X-ray crystallography enabled the optimization to a lead compound that demonstrated favorable potency (IC50 = 2 nM), PK properties, and a robust PK/PD relationship.

A novel glucokinase activator modulates pancreatic islet and hepatocyte function.

The glucose-sensing enzyme glucokinase (GK) plays a key role in glucose metabolism. We report here the effects of a novel glucokinase activator, LY2121260. The activator enhanced GK activity via binding to the allosteric site located in the hinge region of the enzyme. LY2121260 stimulated insulin secretion in a glucose-dependent manner in pancreatic beta-cells and increased glucose use in rat hepatocytes. In addition, incubation of beta-cells with the GK activator resulted in increased GK protein levels, suggesting that enhanced insulin secretion on chronic treatment with a GK activator may be due to not only changed enzyme kinetics but also elevated enzyme levels. Animals treated with LY2121260 showed an improved glucose tolerance after oral glucose challenge. These results support the concept that GK activators represent a new class of compounds that increase both insulin secretion and hepatic glucose use and in doing so may prove to be effective agents for the control of blood glucose levels in patients with type 2 diabetes.

Identification of potent and selective hydantoin inhibitors of aggrecanase-1 and aggrecanase-2 that are efficacious in both chemical and surgical models of osteoarthritis.

A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc metalloproteases commonly referred to as aggrecanase-1 and aggrecanase-2, respectively. These enzymes are involved in the degradation of aggrecan, a key component of cartilage. Inhibitors of these enzymes could be potential osteoarthritis (OA) therapies. A series of hydantoin inhibitors of ADAMTS-4 and ADAMTS-5 were identified from a screening campaign and optimized through structure-based drug design to give hydantoin 13. Hydantoin 13 had excellent selectivity over other zinc metalloproteases such as TACE, MMP2, MMP3, MMP13, and MMP14. The compound also produced efficacy in both a chemically induced and surgical model of OA in rats.

Discovery of Cathepsin S Inhibitor LY3000328 for the Treatment of Abdominal Aortic Aneurysm.

Cathepsin S (Cat S) plays an important role in many pathological conditions, including abdominal aortic aneurysm (AAA). Inhibition of Cat S may provide a new treatment for AAA. To date, several classes of Cat S inhibitors have been reported, many of which form covalent interactions with the active site Cys25. Herein, we report the discovery of a novel series of noncovalent inhibitors of Cat S through a medium-throughput focused cassette screen and the optimization of the resulting hits. Structure-based optimization efforts led to Cat S inhibitors such as 5 and 9 with greatly improved potency and drug disposition properties. This series of compounds binds to the S2 and S3 subsites without interacting with the active site Cys25. On the basis of in vitro potency, selectivity, and efficacy in a CaCl2-induced AAA in vivo model, 5 (LY3000328) was selected for clinical development.

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 5: Combined A- and C-ring structure-activity relationship studies.

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER subtypes alpha and beta in opposite orientations. Here we describe the synthesis of a late stage intermediate that allowed us to combine A-ring and C-ring modifications and carry out simultaneous SAR studies at both positions. Modification of both positions proved additive, maintaining affinity and improving ERbeta selectivity up to 83-fold. An X-ray cocrystal structure confirms the previously observed binding mode in ERbeta.

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 2: structure-activity relationship studies on the benzopyran scaffold.

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER subtypes alpha and beta in opposite orientations. Here we describe structure-activity relationship studies that led to the discovery of bezopyran 5b. X-ray crystal structures of 5b and a non-selective analog 5c in ERalpha help explain the observed selectivity of the benzopyran platform.

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 3: synthesis of cyclopentanone and cyclohexanone intermediates for C-ring modification.

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER subtypes alpha and beta in opposite orientations. Here we describe the syntheses of cyclopentanone and cyclohexanone intermediates for SAR studies of the C-ring on the benzopyran scaffold. Modification of the C-ring disrupts binding to ERalpha, thus improving ERbeta selectivity up to 100-fold. X-ray cocrystal structures confirm previously observed binding modes.

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 4: functionalization of the benzopyran A-ring.

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER receptor subtypes alpha and beta in opposite orientations. We have used structure based drug design to show that this unique phenomena can be exploited via substitution at the 8-position of the benzopyran A-ring to disrupt binding to ERalpha, thus improving ERbeta subtype selectivity. X-ray cocrystal structures with ERalpha and ERbeta are supportive of this approach to improve selectivity in this structural class.