Antibody-drug conjugates (ADCs) combine the selectivity of antibodies with the cytotoxicity of drug payloads to yield highly targeted and potent therapeutics. Owing to the need to chemically modify residues for attachment of the payload and their more complex structure compared to either component alone, ADCs can present additional challenges related to stability of the final drug product. Here, we report for the first time the use of high-throughput experimental screens and computational techniques to tune the conformational and colloidal behavior of a monomethyl auristatin F-based ADC. The ADC, which exhibits high opalescence with strongly attractive protein-protein interactions, is transformed into a more stable structure by experimentally traversing a library of more than ∼100 formulations. A significant reduction in turbidity and increase in diffusion interaction parameter is observed by varying properties such as pH and ionic strength. Computational modeling rationalized these changes and pointed to the presence of attractive electrostatic interactions between ADC molecules facilitated by the drug payload and histidine residues. Taken together, the experimental and computational work presented provides a general roadmap of studies to perform during ADC development to find stable formulations, while the mechanistic learnings can be applied towards the design and stabilization of other IgG1-based ADCs.

We investigated the effects of endothelin-1 on pressor responses to norepinephrine in perfused mesenteric arteries of deoxycorticosterone acetate (DOCA)–salt hypertensive rats. The response to norepinephrine (10−6 M) was significantly increased in DOCA–salt rats compared with that in uninephrectomized control rats. Perfusion of the arteries with subpressor dose of endothelin-1 (3×10−10 M) for 15 min markedly enhanced the pressor responses to norepinephrine (10−6 and 3×10−6 M) in control rats and this effect was significantly prevented by BQ788 [N-cis-2,6-dimethylpiperidinocarbonyl-l-γ-methylleucyl-d-1-methoxycarbonyl-tryptophanyl-d-norleucine] (10−6 M), but not by FR139317 ((R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-pyridyl)propionic acid) (10−6 M). In DOCA–salt hypertensive rats, increased pressor response to norepinephrine was declined to the level of control rats by BQ788, but not by FR139317. In contrast to the case seen with control rat, exogenous endothelin-1 had little effect on pressor responses to norepinephrine in the arteries of DOCA–salt hypertensive rats. Total immunoreactive endothelin content in the arteries of DOCA–salt hypertensive rats was significantly higher than that of uninephrectomized control rats. These results suggest that endothelin-1 enhances contractile responses to norepinephrine through endothelin ETB receptor. Moreover, this phenomenon is stimulated tonically by endogenous endothelin-1 in DOCA–salt hypertensive rats and may contribute to the maintenance of hypertension in DOCA–salt rats.

Biopharmaceutical product and process development do not yet take advantage of predictive computational modeling to nearly the degree seen in industries based on smaller molecules. To assess and advance progress in this area, spirited coopetition (mutually beneficial collaboration between competitors) was successfully used to motivate industrial scientists to develop, share, and compare data and methods which would normally have remained confidential. The first "Highland Games" competition was held in conjunction with the October 2018 Recovery of Biological Products Conference in Ashville, NC, with the goal of benchmarking and assessment of the ability to predict development-related properties of six antibodies from their amino acid sequences alone. Predictions included purification-influencing properties such as isoelectric point and protein A elution pH, and biophysical properties such as stability and viscosity at very high concentrations. Essential contributions were made by a large variety of individuals, including companies which consented to provide antibody amino acid sequences and test materials, volunteers who undertook the preparation and experimental characterization of these materials, and prediction teams who attempted to predict antibody properties from sequence alone. Best practices were identified and shared, and areas in which the community excels at making predictions were identified, as well as areas presenting opportunities for considerable improvement. Predictions of isoelectric point and protein A elution pH were especially good with all-prediction average errors of 0.2 and 1.6 pH unit, respectively, while predictions of some other properties were notably less good. This manuscript presents the events, methods, and results of the competition, and can serve as a tutorial and as a reference for in-house benchmarking by others. Organizations vary in their policies concerning disclosure of methods, but most managements were very cooperative with the Highland Games exercise, and considerable insight into common and best practices is available from the contributed methods. The accumulated data set will serve as a benchmarking tool for further development of in silico prediction tools.

Abelson kinase (c-Abl) is a ubiquitously expressed, nonreceptor tyrosine kinase which plays a key role in cell differentiation and survival. It was hypothesized that transient activation of c-Abl kinase via displacement of the N-terminal autoinhibitory "myristoyl latch", may lead to an increased hematopoietic stem cell differentiation. This would increase the numbers of circulating neutrophils and so be an effective treatment for chemotherapy-induced neutropenia. This paper describes the discovery and optimization of a thiazole series of novel small molecule c-Abl activators, initially identified by a high throughput screening. Subsequently, a scaffold-hop, which exploited the improved physicochemical properties of a dihydropyrazole analogue, identified through fragment screening, delivered potent, soluble, cell-active c-Abl activators, which demonstrated the intracellular activation of c-Abl in vivo.

Bone Morphogenetic Protein 1 (BMP1) inhibition is a potential method for treating fibrosis because BMP1, a member of the zinc metalloprotease family, is required to convert pro-collagen to collagen. A novel class of reverse hydroxamate BMP1 inhibitors was discovered, and cocrystal structures with BMP1 were obtained. The observed binding mode is unique in that the small molecule occupies the nonprime side of the metalloprotease pocket providing an opportunity to build in metalloprotease selectivity. Structure-guided modification of the initial hit led to the identification of an oral in vivo tool compound with selectivity over other metalloproteases. Due to irreversible inhibition of cytochrome P450 3A4 for this chemical class, the risk of potential drug-drug interactions was managed by optimizing the series for subcutaneous injection.

Heterogeneity of biopharmaceutical products is common due to various co- and post-translational modifications and degradation events that occur during the biological production process and throughout the shelf life. Product-related variants resulting from these modifications potentially affect a product's biological activity and safety, and thus, their detailed structure characterization is of great importance for successful development of protein therapeutics. Specifically, in this study, two novel low-level product variants in a recombinant therapeutic protein were characterized via chromatographic enrichment followed by proteolytic digestion and analysis using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). One of the variants was identified to be the therapeutic protein missing a 61-amino-acid fragment from its N-terminus. Consequently, the other variant was found to be the therapeutic protein carrying the 61-amino-acid long peptide. Furthermore, detailed structure at the modification site of the latter variant was determined as that amino group from the protein's N-terminus linked to side chain carbonyl carbon at Asp 61 residue of the peptide, based on the complementary information from collision induced dissociation and electron transfer dissociation MS/MS analysis. Results from sedimentation velocity analytical ultracentrifugation and computational structural modeling supported the hypothesis that formation of these two variants was a result of protein self-association. In dimeric state, the head-to-toe stacking conformation of two therapeutic protein molecules allowed spatial closeness between the N-terminus of one molecule and the 61st amino acid of the other molecule, resulting in a novel peptide transfer  between the two protein molecules.

Over the last decade, a number of computational methods have been developed, which attempt to evaluate the thermodynamic properties of individual water molecules at the solute-solvent interface, in order to assess contributions to protein-ligand binding. In some cases, these tools tell us what we already know, e.g. that hydrophobic pockets prefer lipophilic substituents, and in other cases the methods only seem to add clarity when retrospectively applied. Hence we have grappled with how to utilize such approaches to understand non-intuitive results and to generate chemistry ideas that otherwise would not have been developed. Here we provide our perspective on these methods and describe how results have been interpreted and applied. We include examples from GSK and elsewhere that highlight how water methods have been (1) utilized retrospectively to explain non-intuitive structure- activity relationships and (2) applied prospectively for chemistry design. Finally, we discuss where this field of study could lead to maximal impact in drug discovery research.

c-Abl kinase is maintained in its normal inactive state in the cell through an assembled, compact conformation. We describe two chemical series that bind to the myristoyl site of the c-Abl kinase domain and stimulate c-Abl activation. We hypothesize that these molecules activate c-Abl either by blocking the C-terminal helix from adopting a bent conformation that is critical for the formation of the autoinhibited conformation or by simply providing no stabilizing interactions to the bent conformation of this helix. Structure-based molecular modeling guided the optimization of binding and activation of c-Abl of these two chemical series and led to the discovery of c-Abl activators with nanomolar potency. The small molecule c-Abl activators reported herein could be used as molecular tools to investigate the biological functions of c-Abl and therapeutic implications of its activation.

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.

Small ubiquitin-like modifier (SUMO) belongs to the family of ubiquitin-like proteins (Ubls) that can be reversibly conjugated to target-specific lysines on substrate proteins. Although covalently sumoylated products are readily detectible in gel-based assays, there has been little progress toward the development of robust quantitative sumoylation assay formats for the evaluation of large compound libraries. In an effort to identify inhibitors of ubiquitin carrier protein 9 (Ubc9)-dependent sumoylation, a high-throughput fluorescence polarization assay was developed, which allows detection of Lys-1201 sumoylation, corresponding to the major site of functional sumoylation within the transcriptional repressor trichorhino-phalangeal syndrome type I protein (TRPS1). A minimal hexapeptide substrate peptide, TMR-VVK₁₂₀₁TEK, was used in this assay format to afford high-throughput screening of the GlaxoSmithKline diversity compound collection. A total of 728 hits were confirmed but no specific noncovalent inhibitors of Ubc9 dependent trans-sumoylation were found. However, several diaminopyrimidine compounds were identified as inhibitors in the assay with IC₅₀ values of 12.5 μM. These were further characterized to be competent substrates which were subject to sumoylation by SUMO-Ubc9 and which were competitive with the sumoylation of the TRPS1 peptide substrates.

A series of biarylsulfonamides was identified as hCCR2 receptor antagonist but suffered from high plasma protein binding resulting in a >100 fold shift in activity in a functional GTPγS assay run in tandem in the presence and absence of human serum albumin. Introduction of an aryl amide with ethylenediamine linker led to compounds with reduced shifts and improved activity in whole blood.

c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.