Molecular bottlebrush prodrugs as mono- and triplex combination therapies for multiple myeloma.

Cancer therapies often have narrow therapeutic indexes and involve potentially suboptimal combinations due to the dissimilar physical properties of drug molecules. Nanomedicine platforms could address these challenges, but it remains unclear whether synergistic free-drug ratios translate to nanocarriers and whether nanocarriers with multiple drugs outperform mixtures of single-drug nanocarriers at the same dose. Here we report a bottlebrush prodrug (BPD) platform designed to answer these questions in the context of multiple myeloma therapy. We show that proteasome inhibitor (bortezomib)-based BPD monotherapy slows tumour progression in vivo and that mixtures of bortezomib, pomalidomide and dexamethasone BPDs exhibit in vitro synergistic, additive or antagonistic patterns distinct from their corresponding free-drug counterparts. BPDs carrying a statistical mixture of three drugs in a synergistic ratio outperform the free-drug combination at the same ratio as well as a mixture of single-drug BPDs in the same ratio. Our results address unanswered questions in the field of nanomedicine, offering design principles for combination nanomedicines and strategies for improving current front-line monotherapies and combination therapies for multiple myeloma.

Reply to Correspondence on "Synergy and Antagonism between Allosteric and Active-Site Inhibitors of Abl Tyrosine Kinase".

Manley and co-workers provide data demonstrating that, at super-pharmacological concentrations (300 µM), a ternary complex between Abl, asciminib, and ATP-competitive inhibitors is possible. The work in our manuscript concerns the interplay of asciminib (and GNF-2) with ATP-competitive inhibitors at pharmacologically relevant concentrations (Cmax =1.6-3.7 µM for asciminib). Manley and co-workers do not question any of the studies that we reported, nor do they provide explanations for how our work fits into their preferred model. Herein, we consider the data presented by Manley and co-workers. In addition, we provide new data supporting the findings in our Communication. Asciminib and ATP-competitive inhibitors do not simultaneously bind Abl at pharmacologically relevant concentrations unless the conformation selectivity for both ligands is matched.

Conformation-tunable ATP-competitive kinase inhibitors.

Small molecule kinase inhibitors have shown immense clinical utility for diverse indications. While >60 kinase inhibitors have been approved (and many more in clinical trials), it remains unclear whether the clinical efficacy of a kinase inhibitor is solely dependent on enzymatic inhibition, or whether non-catalytic functions play a role in the efficacy of some kinase inhibitors. Here, we designed and synthesized a series of pyrazolopyrimidine kinase inhibitors that modulate the global kinase conformation of c-Src kinase. Expanding upon our findings from the pyrazolopyrimidine inhibitor series, we designed, synthesized, and evaluated three pair of conformation-selective kinase inhibitors, each with a unique hinge-binding scaffold. We profiled each pair of kinase inhibitors across 468 kinases and identified 38 kinases that could be studied using these pair of conformation-selective inhibitors. We also explore the binding of conformation-selective kinase inhibitors to mutant kinases of EGFR, FLT3, and KIT. Together, these studies yield important insight into the design of conformation-tunable kinase inhibitors and provide a toolset of compounds to study the role of protein conformation on kinase signaling.

Synergy and Antagonism between Allosteric and Active-Site Inhibitors of Abl Tyrosine Kinase.

Allosteric inhibitors of Abl kinase are being explored in the clinic, often in combination with ATP-site inhibitors of Abl kinase. However, there are conflicting data on whether both ATP-competitive inhibitors and myristoyl-site allosteric inhibitors can simultaneously bind Abl kinase. Here, we determine whether there is synergy or antagonism between ATP-competitive inhibitors and allosteric inhibitors of Abl. We observe that clinical ATP-competitive inhibitors are not synergistic with allosteric ABL inhibitors, however, conformation-selective ATP-site inhibitors that modulate the global conformation of Abl can afford synergy. We demonstrate that kinase conformation is the key driver to simultaneously bind two compounds to Abl kinase. Finally, we explore the interaction of allosteric and conformation selective ATP-competitive inhibitors in a series of biochemical and cellular assays.

Anti-MUC1-C Antibody-Conjugated Nanoparticles Potentiate the Efficacy of Fractionated Radiation Therapy.

PURPOSE: Heavy-metal chelators and inorganic nanoparticles (NPs) have been examined as potential radioenhancers to increase the efficacy of external beam radiation therapy for various cancers. Most of these agents have, unfortunately, displayed relatively poor pharmacokinetic properties, which limit the percentage of injected dose (%ID/g) that localizes to tumors and which shorten the window for effective radiation enhancement due to rapid tumor washout. METHODS AND MATERIALS: To address these challenges, we sought to conjugate gadolinium-based ultrasmall (<5 nm) NPs to an antibody directed against the oncogenic MUC1-C subunit that is overexpressed on the surface of many different human cancer types. The binding of the anti-MUC1-C antibody 3D1 to MUC1-C on the surface of a cancer cell is associated with its internalization and, thereby, to effective intracellular delivery of the antibody-associated payload, promoting its effective tumor retention. As such, we examined whether systemically administered anti-MUC1-C antibody-conjugated, gadolinium-based NPs (anti-MUC1-C/NPs) could accumulate within cell-line xenograft models of MUC1-C-expressing (H460) lung and (E0771) breast cancers to improve the efficacy of radiation therapy (XRT). RESULTS: The %ID/g of anti-MUC1-C/NPs that accumulated within tumors was found to be similar to that of their unconjugated counterparts (6.6 ± 1.4 vs 5.9 ± 1.7 %ID/g, respectively). Importantly, the anti-MUC1-C/NPs demonstrated prolonged retention in in vivo tumor microenvironments; as a result, the radiation boost was maintained during the course of fractionated therapy (3 × 5.2 Gy). We found that by administering anti-MUC1-C/NPs with XRT, it was possible to significantly augment tumor growth inhibition and to prolong the animals' overall survival (46.2 ± 3.1 days) compared with the administration of control NPs with XRT (31.1 ± 2.4 days) or with XRT alone (27.3 ± 1.6 days; P < .01, log-rank). CONCLUSIONS: These findings suggest that anti-MUC1-C/NPs could be used to enhance the effectiveness of radiation therapy and potentially to improve clinical outcomes.

Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma.

Precursor states of Multiple Myeloma (MM) and its native tumor microenvironment need in-depth molecular characterization to better stratify and treat patients at risk. Using single-cell RNA sequencing of bone marrow cells from precursor stages, MGUS and smoldering myeloma (SMM), to full-blown MM alongside healthy donors, we demonstrate early immune changes during patient progression. We find NK cell abundance is frequently increased in early stages, and associated with altered chemokine receptor expression. As early as SMM, we show loss of GrK+ memory cytotoxic T-cells, and show their critical role in MM immunosurveillance in mouse models. Finally, we report MHC class II dysregulation in CD14+ monocytes, which results in T cell suppression in vitro. These results provide a comprehensive map of immune changes at play over the evolution of pre-malignant MM, which will help develop strategies for immune-based patient stratification.

Selective Proteolysis to Study the Global Conformation and Regulatory Mechanisms of c-Src Kinase.

Protein kinase pathways are traditionally mapped by monitoring downstream phosphorylation. Meanwhile, the noncatalytic functions of protein kinases remain under-appreciated as critical components of kinase signaling. c-Src is a protein kinase known to have noncatalytic signaling function important in healthy and disease cell signaling. Large conformational changes in the regulatory domains regulate c-Src's noncatalytic functions. Herein, we demonstrate that changes in the global conformation of c-Src can be monitored using a selective proteolysis methodology. Further, we use this methodology to investigate changes in the global conformation of several clinical and nonclinical mutations of c-Src. Significantly, we identify a novel activating mutation observed clinically, W121R, that can escape down-regulation mechanisms. Our methodology can be expanded to monitor the global conformation of other tyrosine kinases, including c-Abl, and represents an important tool toward the elucidation of the noncatalytic functions of protein kinases.

Modulating noncatalytic function with kinase inhibitors.

In this issue of Chemistry & Biology, Hari and colleagues show that conformation-selective ATP-competitive kinase inhibitors have distinct noncatalytic effects on Erk2, including the ability to modulate protein-protein interactions outside the ATP-binding site. These findings enhance our knowledge about the diverse array of activities in which kinase inhibitors can target signaling pathways.

X-ray structural and biological evaluation of a series of potent and highly selective inhibitors of human coronavirus papain-like proteases.

Structure-guided design was used to generate a series of noncovalent inhibitors with nanomolar potency against the papain-like protease (PLpro) from the SARS coronavirus (CoV). A number of inhibitors exhibit antiviral activity against SARS-CoV infected Vero E6 cells and broadened specificity toward the homologous PLP2 enzyme from the human coronavirus NL63. Selectivity and cytotoxicity studies established a more than 100-fold preference for the coronaviral enzyme over homologous human deubiquitinating enzymes (DUBs), and no significant cytotoxicity in Vero E6 and HEK293 cell lines is observed. X-ray structural analyses of inhibitor-bound crystal structures revealed subtle differences between binding modes of the initial benzodioxolane lead (15g) and the most potent analogues 3k and 3j, featuring a monofluoro substitution at para and meta positions of the benzyl ring, respectively. Finally, the less lipophilic bis(amide) 3e and methoxypyridine 5c exhibit significantly improved metabolic stability and are viable candidates for advancing to in vivo studies.