Scott E. Warder

Reducing agent-mediated precipitation of high-abundance plasma proteins

Depletion of high-abundance proteins is regarded as a critical sample preparation step for most plasma proteomic analyses and profiling strategies. This report describes a process that rapidly and reproducibly precipitates high-abundance disulfide-rich proteins, including albumin and transferrin, from serum and plasma. A low volume of concentrated reducing agent, viz. dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), was added directly to plasma followed by a brief incubation at ambient temperature. Removal of the precipitate via centrifugation and identification of the protein content revealed an albumin-enriched pellet. Direct analysis of the supernatant by MALDI-TOF-MS afforded peptidome and small protein profiles with enhanced features and minimal ionization of full-length albumin. The reproducible and quantitative nature of the method has been demonstrated by monitoring the plasma levels of an antiangiogenic protein biologic, rKringle5 (rK5). The 10.5-kDa analyte was only reliably detected in plasma after treatment with reducing agent, ionizing linearly from 150 to 1200 fmol (on-target) with a mean CV of 7%. This method distinguishes itself from immunoaffinity resin-based approaches since it can be scaled to large milliliter quantities and it is compatible with plasma from all species tested.

Bcl-XL represents a druggable molecular vulnerability during aurora B inhibitor-mediated polyploidization.

Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This “addiction” can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility.

Gene Expression Analysis in Rats Treated with Experimental Acetyl-Coenzyme A Carboxylase Inhibitors Suggests Interactions with the Peroxisome Proliferator-Activated Receptor α Pathway

Acetyl CoA carboxylase (ACC) 2, which catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, has been identified as a potential target for type 2 diabetes and obesity. Small-molecule inhibitors of ACC2 would be expected to reduce de novo lipid synthesis and increase lipid oxidation. Treatment of ob/ob mice with compound A-908292 (S) ({(S)-3-[2-(4-isopropoxy-phenoxy)-thiazol-5-yl]-1-methyl-prop-2-ynyl}-carbamic acid methyl ester), a small-molecule inhibitor with an IC50 of 23 nM against ACC2, resulted in a reduction of serum glucose and triglyceride levels. However, compound A-875400 (R) ({(R)-3-[2-(4-isopropoxy-phenoxy)-thiazol-5-yl]-1-methyl-prop-2-ynyl}-carbamic acid methyl ester), an inactive enantiomer of A-908292 (S) with approximately 50-fold less activity against ACC2, also caused a similar reduction in glucose and triglycerides, suggesting that the glucose-lowering effects in ob/ob mice may be mediated by other metabolic pathways independent of ACC2 inhibition. To characterize the pharmacological activity of these experimental compounds at a transcriptional level, rats were orally dosed for 3 days with either A-908292 (S) or A-875400 (R), and gene expression analysis was performed. Gene expression analysis of livers showed that treatment with A-908292 (S) or A-875400 (R) resulted in gene expression profiles highly similar to known peroxisome proliferator-activated receptor (PPAR)-α activators. The results suggest that, in vivo, both A-908292 (S) and A-875400 (R) stimulated the PPAR-α-dependent signaling pathway. These results were further supported by both an in vitro genomic evaluation using rat hepatocytes and immunohistochemical evaluation using 70-kDa peroxisomal membrane protein. Overall, the gene expression analysis suggests a plausible mechanism for the similar pharmacological findings with active and inactive enantiomers of an ACC2 inhibitor.

Fragment-Based, Structure-Enabled Discovery of Novel Pyridones and Pyridone Macrocycles as Potent Bromodomain and Extra-Terminal Domain (BET) Family Bromodomain Inhibitors

Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 μM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.

Preclinical Characterization of BET Family Bromodomain Inhibitor ABBV-075 Suggests Combination Therapeutic Strategies

ABBV-075 is a potent and selective BET family bromodomain inhibitor that recently entered phase I clinical trials. Comprehensive preclinical characterization of ABBV-075 demonstrated broad activity across cell lines and tumor models, representing a variety of hematologic malignancies and solid tumor indications. In most cancer cell lines derived from solid tumors, ABBV-075 triggers prominent G1 cell-cycle arrest without extensive apoptosis. In this study, we show that ABBV-075 efficiently triggers apoptosis in acute myeloid leukemia (AML), non-Hodgkin lymphoma, and multiple myeloma cells. Apoptosis induced by ABBV-075 was mediated in part by modulation of the intrinsic apoptotic pathway, exhibiting synergy with the BCL-2 inhibitor venetoclax in preclinical models of AML. In germinal center diffuse large B-cell lymphoma, BCL-2 levels or venetoclax sensitivity predicted the apoptotic response to ABBV-075 treatment. In vivo combination studies uncovered surprising benefits of low doses of ABBV-075 coupled with bortezomib and azacitidine treatment, despite the lack of in vitro synergy between ABBV-075 and these agents. The in vitro/in vivo activities of ABBV-075 described here may serve as a useful reference to guide the development of ABBV-075 and other BET family inhibitors for cancer therapy. Cancer Res; 77(11); 2976-89. ©2017 AACR.

Target Identification of Compounds from a Cell Viability Phenotypic Screen Using a Bead/Lysate-Based Affinity Capture Platform

The pharmaceutical industry has been continually challenged by dwindling target diversity. To obviate this trend, phenotypic screens have been adopted, complementing target-centric screening approaches. Phenotypic screens identify drug leads using clinically relevant and translatable mechanisms, remaining agnostic to targets. While target anonymity is advantageous early in the drug discovery process, it poses challenges to hit progression, including the development of backup series, retaining desired pharmacology during optimization, discovery of markers, and understanding mechanism-driven toxicity. Consequently, significant effort has been expended to elaborate the targets and mechanisms at work for promising screening hits. Affinity capture is commonly leveraged, where the compounds are linked to beads and targets are abstracted from cell homogenates. This technique has proven effective for identifying targets of kinase, PARP, and HDAC inhibitors, and examples of new targets have been reported. Herein, a three-pronged approach to target deconvolution by affinity capture is described, including the implementation of a uniqueness index that helps discriminate between bona fide targets and background. The effectiveness of this approach is demonstrated using characterized compounds that act on known and noncanonical target classes. The platform is subsequently applied to phenotypic screening hits, identifying candidate targets. The success rate of bead-based affinity capture is discussed.

Discovery of N-(4-(2,4-difluorophenoxy)-3-(6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridin-4-yl)phenyl)ethanesulfonamide (ABBV-075/mivebresib), a Potent and Orally Available Bromodomain and Extraterminal domain (BET) Family Bromodomain Inhibitor

The development of bromodomain and extraterminal domain (BET) bromodomain inhibitors and their examination in clinical studies, particularly in oncology settings, has garnered substantial recent interest. An effort to generate novel BET bromodomain inhibitors with excellent potency and drug metabolism and pharmacokinetics (DMPK) properties was initiated based upon elaboration of a simple pyridone core. Efforts to develop a bidentate interaction with a critical asparagine residue resulted in the incorporation of a pyrrolopyridone core, which improved potency by 9-19-fold. Additional structure-activity relationship (SAR) efforts aimed both at increasing potency and improving pharmacokinetic properties led to the discovery of the clinical candidate 63 (ABBV-075/mivebresib), which demonstrates excellent potency in biochemical and cellular assays, advantageous exposures and half-life both in animal models and in humans, and in vivo efficacy in mouse models of cancer progression and inflammation.

Methylpyrrole inhibitors of BET bromodomains

An NMR fragment screen for binders to the bromodomains of BRD4 identified 2-methyl-3-ketopyrroles 1 and 2. Elaboration of these fragments guided by structure-based design provided lead molecules with significant activity in a mouse tumor model. Further modifications to the methylpyrrole core provided compounds with improved properties and enhanced activity in a mouse model of multiple myeloma.

Screening Strategies and Methods for Better Off-Target Liability Prediction and Identification of Small-Molecule Pharmaceuticals:

Pharmaceutical discovery and development is a long and expensive process that, unfortunately, still results in a low success rate, with drug safety continuing to be a major impedance. Improved safety screening strategies and methods are needed to more effectively fill this critical gap. Recent advances in informatics are now making it possible to manage bigger data sets and integrate multiple sources of screening data in a manner that can potentially improve the selection of higher-quality drug candidates. Integrated screening paradigms have become the norm in Pharma, both in discovery screening and in the identification of off-target toxicity mechanisms during later-stage development. Furthermore, advances in computational methods are making in silico screens more relevant and suggest that they may represent a feasible option for augmenting the current screening paradigm. This paper outlines several fundamental methods of the current drug screening processes across Pharma and emerging techniques/technologies that promise to improve molecule selection. In addition, the authors discuss integrated screening strategies and provide examples of advanced screening paradigms.

Emerging Approaches for the Identification of Protein Targets of Small Molecules - A Practitioners’ Perspective

Small-molecule (SM) leads in the early drug discovery pipeline are progressed primarily based on potency against the intended target(s) and selectivity against a very narrow slice of the proteome. So, why is there a tendency to wait until SMs are matured before probing for a deeper mechanistic understanding? For one, there is a concern about the interpretation of complex -omic data outputs and the resources needed to test these hypotheses. However, with recent advances in broad endpoint profiling assays that have companion reference databases and refined technology integration strategies, we argue that data complexity can translate into meaningful decision-making. This same strategy can also prioritize phenotypic screening hits to increase the likelihood of accessing unprecedented target space. In this Perspective. we will highlight a cohesive process that supports SM hit prosecution, providing a data-driven rationale and a suite of methods for direct identification of SM targets driving relevant biological end points.