Coiled-coil domains are sufficient to drive liquid-liquid phase separation in protein models.

Liquid-liquid phase separation (LLPS) is thought to be a main driving force in the formation of membraneless organelles. Examples of such organelles include the centrosome, central spindle, and stress granules. Recently, it has been shown that coiled-coil (CC) proteins, such as the centrosomal proteins pericentrin, spd-5, and centrosomin, might be capable of LLPS. CC domains have physical features that could make them the drivers of LLPS, but it is unknown if they play a direct role in the process. We developed a coarse-grained simulation framework for investigating the LLPS propensity of CC proteins, in which interactions that support LLPS arise solely from CC domains. We show, using this framework, that the physical features of CC domains are sufficient to drive LLPS of proteins. The framework is specifically designed to investigate how the number of CC domains, as well as the multimerization state of CC domains, can affect LLPS. We show that small model proteins with as few as two CC domains can phase separate. Increasing the number of CC domains up to four per protein can somewhat increase LLPS propensity. We demonstrate that trimer-forming and tetramer-forming CC domains have a dramatically higher LLPS propensity than dimer-forming coils, which shows that multimerization state has a greater effect on LLPS than the number of CC domains per protein. These data support the hypothesis of CC domains as drivers of protein LLPS, and have implications in future studies to identify the LLPS-driving regions of centrosomal and central spindle proteins.

Programmable de novo designed coiled coil-mediated phase separation in mammalian cells.

Membraneless liquid compartments based on phase-separating biopolymers have been observed in diverse cell types and attributed to weak multivalent interactions predominantly based on intrinsically disordered domains. The design of liquid-liquid phase separated (LLPS) condensates based on de novo designed tunable modules that interact in a well-understood, controllable manner could improve our understanding of this phenomenon and enable the introduction of new features. Here we report the construction of CC-LLPS in mammalian cells, based on designed coiled-coil (CC) dimer-forming modules, where the stability of CC pairs, their number, linkers, and sequential arrangement govern the transition between diffuse, liquid and immobile condensates and are corroborated by coarse-grained molecular simulations. Through modular design, we achieve multiple coexisting condensates, chemical regulation of LLPS, condensate fusion, formation from either one or two polypeptide components or LLPS regulation by a third polypeptide chain. These findings provide further insights into the principles underlying LLPS formation and a design platform for controlling biological processes.

Coiled-coil domains are sufficient to drive liquid-liquid phase separation of proteins in molecular models.

Liquid-liquid phase separation (LLPS) is thought to be a main driving force in the formation of membraneless organelles. Examples of such organelles include the centrosome, central spindle, and stress granules. Recently, it has been shown that coiled-coil (CC) proteins, such as the centrosomal proteins pericentrin, spd-5, and centrosomin, might be capable of LLPS. CC domains have physical features that could make them the drivers of LLPS, but it is unknown if they play a direct role in the process. We developed a coarse-grained simulation framework for investigating the LLPS propensity of CC proteins, in which interactions which support LLPS arise solely from CC domains. We show, using this framework, that the physical features of CC domains are sufficient to drive LLPS of proteins. The framework is specifically designed to investigate how the number of CC domains, as well as multimerization state of CC domains, can affect LLPS. We show that small model proteins with as few as two CC domains can phase separate. Increasing the number of CC domains up to four per protein can somewhat increase LLPS propensity. We demonstrate that trimer-forming and tetramer-forming CC domains have a dramatically higher LLPS propensity than dimer-forming coils, which shows that multimerization state has a greater effect on LLPS than the number of CC domains per protein. These data support the hypothesis of CC domains as drivers of protein LLPS, and has implications in future studies to identify the LLPS-driving regions of centrosomal and central spindle proteins.

Vinblastine pharmacokinetics in mouse, dog, and human in the context of a physiologically based model incorporating tissue-specific drug binding, transport, and metabolism.

Vinblastine (VBL) is a vinca alkaloid-class cytotoxic chemotherapeutic that causes microtubule disruption and is typically used to treat hematologic malignancies. VBL is characterized by a narrow therapeutic index, with key dose-limiting toxicities being myelosuppression and neurotoxicity. Pharmacokinetics (PK) of VBL is primarily driven by ABCB1-mediated efflux and CYP3A4 metabolism, creating potential for drug-drug interaction. To characterize sources of variability in VBL PK, we developed a physiologically based pharmacokinetic (PBPK) model in Mdr1a/b(-/-) knockout and wild-type mice by incorporating key drivers of PK, including ABCB1 efflux, CYP3A4 metabolism, and tissue-specific tubulin binding, and scaled this model to accurately simulate VBL PK in humans and pet dogs. To investigate the capability of the model to capture interindividual variability in clinical data, virtual populations of humans and pet dogs were generated through Monte Carlo simulation of physiologic and biochemical parameters and compared to the clinical PK data. This model provides a foundation for predictive modeling of VBL PK. The base PBPK model can be further improved with supplemental experimental data identifying drug-drug interactions, ABCB1 polymorphisms and expression, and other sources of physiologic or metabolic variability.

Identifying signatures of proteolytic stability and monomeric propensity in O-glycosylated insulin using molecular simulation.

Insulin has been commonly adopted as a peptide drug to treat diabetes as it facilitates the uptake of glucose from the blood. The development of oral insulin remains elusive over decades owing to its susceptibility to the enzymes in the gastrointestinal tract and poor permeability through the intestinal epithelium upon dimerization. Recent experimental studies have revealed that certain O-linked glycosylation patterns could enhance insulin's proteolytic stability and reduce its dimerization propensity, but understanding such phenomena at the molecular level is still difficult. To address this challenge, we proposed and tested several structural determinants that could potentially influence insulin's proteolytic stability and dimerization propensity. We used these metrics to assess the properties of interest from [Formula: see text] aggregate molecular dynamics of each of 12 targeted insulin glyco-variants from multiple wild-type crystal structures. We found that glycan-involved hydrogen bonds and glycan-dimer occlusion were useful metrics predicting the proteolytic stability and dimerization propensity of insulin, respectively, as was in part the solvent-accessible surface area of proteolytic sites. However, other plausible metrics were not generally predictive. This work helps better explain how O-linked glycosylation influences the proteolytic stability and monomeric propensity of insulin, illuminating a path towards rational molecular design of insulin glycoforms.

Cannabidiol Induces Apoptosis and Perturbs Mitochondrial Function in Human and Canine Glioma Cells.

Cannabidiol (CBD), the major non-psychoactive compound found in cannabis, is frequently used both as a nutraceutical and therapeutic. Despite anecdotal evidence as an anticancer agent, little is known about the effect CBD has on cancer cells. Given the intractability and poor prognoses of brain cancers in human and veterinary medicine, we sought to characterize the in vitro cytotoxicity of CBD on human and canine gliomas. Glioma cells treated with CBD showed a range of cytotoxicity from 4.9 to 8.2 µg/ml; canine cells appeared to be more sensitive than human. Treatment with >5 µg/ml CBD invariably produced large cytosolic vesicles. The mode of cell death was then interrogated using pharmacologic inhibitors. Inhibition of apoptosis was sufficient to rescue CBD-mediated cytotoxicity. Inhibition of RIPK3, a classical necroptosis kinase, also rescued cells from death and prevented the formation of the large cytosolic vesicles. Next, cellular mitochondrial activity in the presence of CBD was assessed and within 2 hours of treatment CBD reduced oxygen consumption in a dose dependent manner with almost complete ablation of activity at 10 µg/ml CBD. Fluorescent imaging with a mitochondrial-specific dye revealed that the large cytosolic vesicles were, in fact, swollen mitochondria. Lastly, calcium channels were pharmacologically inhibited and the effect on cell death was determined. Inhibition of mitochondrial channel VDAC1, but not the TRPV1 channel, rescued cells from CBD-mediated cytotoxicity. These results demonstrate the cytotoxic nature of CBD in human and canine glioma cells and suggest a mechanism of action involving dysregulation of calcium homeostasis and mitochondrial activity.

First-in-Class Inhibitors of Oncogenic CHD1L with Preclinical Activity against Colorectal Cancer.

Since the discovery of CHD1L in 2008, it has emerged as an oncogene implicated in the pathology and poor prognosis of a variety of cancers, including gastrointestinal cancers. However, a mechanistic understanding of CHD1L as a driver of colorectal cancer has been limited. Until now, there have been no reported inhibitors of CHD1L, also limiting its development as a molecular target. We sought to characterize the clinicopathologic link between CHD1L and colorectal cancer, determine the mechanism(s) by which CHD1L drives malignant colorectal cancer, and discover the first inhibitors with potential for novel treatments for colorectal cancer. The clinicopathologic characteristics associated with CHD1L expression were evaluated using microarray data from 585 patients with colorectal cancer. Further analysis of microarray data indicated that CHD1L may function through the Wnt/TCF pathway. Thus, we conducted knockdown and overexpression studies with CHD1L to determine its role in Wnt/TCF-driven epithelial-to-mesenchymal transition (EMT). We performed high-throughput screening (HTS) to identify the first CHD1L inhibitors. The mechanism of action, antitumor efficacy, and drug-like properties of lead CHD1L inhibitors were determined using biochemical assays, cell models, tumor organoids, patient-derived tumor organoids, and in vivo pharmacokinetics and pharmacodynamics. Lead CHD1L inhibitors display potent in vitro antitumor activity by reversing TCF-driven EMT. The best lead CHD1L inhibitor possesses drug-like properties in pharmacokinetic/pharmacodynamic mouse models. This work validates CHD1L as a druggable target and establishes a novel therapeutic strategy for the treatment of colorectal cancer.

Drug Design Targeting T-Cell Factor-Driven Epithelial-Mesenchymal Transition as a Therapeutic Strategy for Colorectal Cancer.

Metastasis is the cause of 90% of mortality in cancer patients. For metastatic colorectal cancer (mCRC), the standard-of-care drug therapies only palliate the symptoms but are ineffective, evidenced by a low survival rate of ∼11%. T-cell factor (TCF) transcription is a major driving force in CRC, and we have characterized it to be a master regulator of epithelial-mesenchymal transition (EMT). EMT transforms relatively benign epithelial tumor cells into quasi-mesenchymal or mesenchymal cells that possess cancer stem cell properties, promoting multidrug resistance and metastasis. We have identified topoisomerase IIα (TOP2A) as a DNA-binding factor required for TCF-transcription. Herein, we describe the design, synthesis, biological evaluation, and in vitro and in vivo pharmacokinetic analysis of TOP2A ATP-competitive inhibitors that prevent TCF-transcription and modulate or reverse EMT in mCRC. Unlike TOP2A poisons, ATP-competitive inhibitors do not damage DNA, potentially limiting adverse effects. This work demonstrates a new therapeutic strategy targeting TOP2A for the treatment of mCRC and potentially other types of cancers.

Kinetics of Cyclophosphamide Metabolism in Humans, Dogs, Cats, and Mice and Relationship to Cytotoxic Activity and Pharmacokinetics.

Cyclophosphamide (CP), a prodrug that is enzymatically converted to the cytotoxic 4-hydroxycyclophosphamide (4OHCP) by hepatic enzymes, is commonly used in both human and veterinary medicine to treat cancers and modulate the immune system. We investigated the metabolism of CP in humans, dogs, cats, and mice using liver microsomes; apparent K M, V max, and intrinsic clearance (V max/K M) parameters were estimated. The interspecies and intraspecies variations in kinetics were vast. Dog microsomes were, on average, 55-fold more efficient than human microsomes, 2.8-fold more efficient than cat microsomes, and 1.2-fold more efficient than mouse microsomes at catalyzing CP bioactivation. These differences translated to cell-based systems. Breast cancer cells exposed to 4OHCP via CP bioactivation by microsomes resulted in a stratification of cytotoxicity that was dependent on the species of microsomes measured by IC50: dog (31.65 µM), mouse (44.95 µM), cat (272.6 µM), and human (1857 µM). The contributions of cytochrome P450s, specifically, CYP2B, CYP2C, and CYP3A, to CP bioactivation were examined: CYP3A inhibition resulted in no change in 4OHCP formation; CYP2B inhibition slightly reduced 4OHCP in humans, cats, and mice; and CYP2C inhibition drastically reduced 4OHCP formation in each species. Semiphysiologic modeling of CP metabolism using scaled metabolic parameters resulted in simulated data that closely matched published pharmacokinetic profiles, determined by noncompartmental analysis. The results highlight differential CP metabolism delineated by species and demonstrate the importance of metabolism on CP clearance.