Inhibition of mutationally activated HER2.

Human epidermal growth factor receptor 2 (HER2) is an oncogenic driver and key therapeutic target for human cancers. Current therapies targeting HER2 are primarily based on overexpression of the wild-type form of HER2. However, kinase domain mutations have been identified that can increase the activity of HER2 even when expressed at basal levels. Using purified enzymes, we confirmed the hyperactivity of two HER2 mutants (D769Y and P780insGSP). To identify small molecule inhibitors against these cancer-associated variants, we used a combined approach consisting of biochemical testing, similarity-based searching, and in silico modeling. These approaches resulted in the identification of a candidate molecule that inhibits mutant forms of HER2 in vitro and in cell-based assays. Our structural model predicts that the compound takes advantage of water-mediated interactions in the HER2 kinase binding pocket.

INK4 Tumor Suppressor Proteins Mediate Resistance to CDK4/6 Kinase Inhibitors.

Cyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C-cyclin D-CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance. SIGNIFICANCE: CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6-INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases.This article is highlighted in the In This Issue feature, p. 275.

Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance.

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type-like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

Identification of a Water-Coordinating HER2 Inhibitor by Virtual Screening Using Similarity-Based Scoring.

Human epidermal growth factor receptor 2 (HER2) is a validated breast cancer drug target for small molecule inhibitors that target the ATP-binding pocket of the kinase domain. In this work, a large-scale virtual screen was performed to a novel homology model of HER2, in a hypothesized "fully active" state, that considered water-mediated interactions during the prioritization of compounds for experimental testing. This screen led to the identification of a new inhibitor with micro molar affinity and potency ( Kd = 7.0 µM, IC50 = 4.6 µM). Accompanying molecular dynamics simulations showed that inhibitor binding likely involves water coordination through an important water-mediated network previously identified in our laboratory. The predicted binding geometry also showed a remarkable overlap with the crystallographic poses for two previously reported inhibitors of the related Chk1 kinase. Concurrent with the HER2 studies, we developed formalized computational protocols that leverage solvated footprints (per-residue interaction maps that include bridging waters) to identify ligands that can "coordinate" or "displace" key binding site waters. Proof-of-concept screens targeting HIVPR and PARP1 demonstrate that molecules with high footprint overlap can be effectively identified in terms of their coordination or displacement patterns relative to a known reference. Overall, the procedures developed as a result of this study should be useful for researchers targeting HER2 and, more generally, for any protein in which the identification of compounds that exploit binding site waters is desirable.

Describing Myxococcus xanthus aggregation using Ostwald ripening equations for thin liquid films.

When starved, a swarm of millions of Myxococcus xanthus cells coordinate their movement from outward swarming to inward coalescence. The cells then execute a synchronous program of multicellular development, arranging themselves into dome shaped aggregates. Over the course of development, about half of the initial aggregates disappear, while others persist and mature into fruiting bodies. This work seeks to develop a quantitative model for aggregation that accurately simulates which will disappear and which will persist. We analyzed time-lapse movies of M. xanthus development, modeled aggregation using the equations that describe Ostwald ripening of droplets in thin liquid films, and predicted the disappearance and persistence of aggregates with an average accuracy of 85%. We then experimentally validated a prediction that is fundamental to this model by tracking individual fluorescent cells as they moved between aggregates and demonstrating that cell movement towards and away from aggregates correlates with aggregate disappearance. Describing development through this model may limit the number and type of molecular genetic signals needed to complete M. xanthus development, and it provides numerous additional testable predictions.