Exploration of macrocyclic peptide binders to the extracellular CRD domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

Elevated levels of receptor tyrosine kinase-like orphan receptor 1 (RORl) expression are observed in multiple hematological and solid tumors, but not in most of the healthy adult tissues, identifying ROR1 as an attractive target for tumor-specific therapy. Herein we will describe the discovery of macrocyclic peptides as binders of the extracellular Cysteine-Rich Domain (CRD) of human ROR1 via mRNA in vitro selection technology using the PDPS platform, followed by exploration of sidechain SAR of parent macrocycle peptides, fluorescently labeled analogs, and a Peptide Drug Conjugate (PDC). The parent macrocyclic peptides represented by Compound 1 and Compound 14 displayed nanomolar cell-based binding to ROR1 and relatively good internalization in 786-O and MDA-MB-231 tumor cell lines. However, these peptides were not observed to induce apoptosis in Mia PaCa-2 cells, a model pancreatic tumor cell line with a relatively low level of cell surface expression of ROR1.

Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail.

Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.

Advancing pharmacy technician training and practice models in the United States: Historical perspectives, workforce development needs, and future opportunities.

The United States healthcare system faces immense challenges related to cost, quality, and access. As the pharmacy profession addresses these challenges by shifting toward a practice model centered around direct patient care clinical services, a competent and capable technician workforce is needed to support the roles of pharmacists. Until recently, little focus has been paid to pharmacy technicians or their role as they relate to practice model change. With ongoing pharmacist practice transformation, an approach that ensures uniform technician education, training, registration, and certification is vital to support a practice model designed to transform medication management across the continuum of care. The purpose of this commentary is three-fold: to review the history of pharmacy technician training and practice, discuss current and future technician practice models, and examine workforce development implications.

A Comparison of Approaches to Student Pharmacist Business Planning in Pharmacy Practice Management.

Objective. To compare second-year student pharmacists' perspectives on two approaches for completing a pharmacy practice management business planning project. Methods. A mixed-methods approach was used to compare two options (traditional and experimental) for completing business plan projects that were offered to teams of second-year student pharmacists as part of a required pharmacy management course. Teams who chose the traditional project were required to develop a unique, pharmacy-related business plan while those who chose the experimental concept were paired with a pharmacy-focused firm within Tennessee and tasked with designing a potential service for the firm's consideration. At semester's end, all students were asked to complete a brief survey to provide insight on their experiences with either of the group projects. Students and firm stakeholders were also asked to participate in a group and individual interview, respectively. Results. Student group comparisons indicated that the experimental project provided a more real-world, business planning experience. Additionally, groups that did the experimental project were more likely to report seeing how business and pharmacy practice were connected, indicate a better understanding of the principles of pharmacy management, and be perceived as more marketable for a future pharmacy career. Firm representatives indicated that insight provided by the students was valuable and that they had plans to implement what was proposed. Conclusion. Connecting student pharmacists with a pharmacy-focused firm provided a real-world management experience that better complemented the course's principles, and created a mutually beneficial innovation-focused partnership.

A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses.

A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 µM relative to IC50 values of 28 to 73 µM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment.

Development of a Human Whole Blood Screening Platform to Monitor JAK/STAT Signaling Using High-Throughput Flow Cytometry.

Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the automation technologies and process optimizations developed here overcame sample integrity, handling, and multiparametric data analysis bottlenecks without affecting assay performance. Together these efforts dramatically increased sample throughput compared to conventional manual flow cytometric approaches and enabled development of novel JAK/STAT inhibitors.

Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin.

Recent evidence indicates that newly synthesized membrane proteins that share the same distributions in the plasma membranes of polarized epithelial cells can pursue a variety of distinct trafficking routes as they travel from the Golgi complex to their common destination at the cell surface. In most polarized epithelial cells, both the Na,K-ATPase and E-cadherin are localized to the basolateral domains of the plasma membrane. To examine the itineraries pursued by newly synthesized Na,K-ATPase and E-cadherin in polarized MDCK epithelial cells, we used the SNAP and CLIP labeling systems to fluorescently tag temporally defined cohorts of these proteins and observe their behaviors simultaneously as they traverse the secretory pathway. These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase. Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19 °C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network. Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking.

SNAP-tag to monitor trafficking of membrane proteins in polarized epithelial cells.

In order to understand the mechanisms through which apical and basolateral membrane proteins achieve their subcellular distributions in polarized epithelial cells, it is critical to develop techniques that permit the selective observation of newly synthesized populations of these proteins. The SNAP tag system permits the detection and visualization of distinct spatially and temporally defined cohorts of tagged proteins. Thus, this technique is especially well suited to studying the trafficking routes pursued by newly synthesized proteins. The SNAP tag can be applied in the setting of fixed or live cell fluorescence microscopic analysis and can also be used in the context of various biochemical approaches. Here, we describe the use of the SNAP tag in association with confocal microscopy and SDS-PAGE to follow the biosynthetic pool of a membrane protein as it exits from the trans-Golgi network and makes its way to the plasma membrane.

AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression.

The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.

Association with {beta}-COP regulates the trafficking of the newly synthesized Na,K-ATPase.

Plasma membrane expression of the Na,K-ATPase requires assembly of its α- and ß-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase α-subunit and the coat protein, ß-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase ß-subunit, the Na,K-ATPase α-subunit interacts with ß-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase ß-subunit, the α-subunit does not interact with ß-COP and traffics to the plasma membrane. Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase α- and ß-subunits, newly synthesized α-subunit associates with ß-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the α- and ß-subunits to form the pump holoenzyme. The interaction with ß-COP was reduced by mutating a dibasic motif at Lys(54) in the Na,K-ATPase α-subunit. This mutant α-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase ß-subunit expression. Although the Lys(54) α-subunit reaches the cell surface without need for ß-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the ß-subunit.

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells.

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse-chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.

VP2 cleavage and the leucine ring at the base of the fivefold cylinder control pH-dependent externalization of both the VP1 N terminus and the genome of minute virus of mice.

Cylindrical projections surrounding the fivefold-symmetry axes in minute virus of mice (MVM) harbor central pores that penetrate through the virion shell. In newly released DNA-containing particles, these pores contain residues 28 to 38 belonging to a single copy of VP2, disposed so that its extreme N-terminal domain projects outside the particle. Virions are metastable, initially sequestering internally the N termini of all copies of the minor capsid protein, VP1, that is essential for entry. This VP1 domain can be externalized in vitro in response to limited heating, and we show here that the efficiency of this transition is greatly enhanced by proteolysis of VP2 N termini to yield VP3. This step also renders the VP1 rearrangement pH dependent, indicating that VP2 cleavage is a maturation step required to prime subsequent emergence of the VP1 "entry" domain. The tightest constriction within the cylinder is created by VP2 leucine 172, the five symmetry-related copies of which form a portal that resembles an iris diaphragm across the base of the pore. In MVMp, threonine substitution at this position, L172T, yields infectious particles following transfection at 37 degrees C, but these can initiate infection only at 32 degrees C, and this process can be blocked by exposing virions to a cellular factor(s) at 37 degrees C during the first 8 h after entry. At 32 degrees C, the mutant particle is highly infectious, and it remains stable prior to VP2 cleavage or following cleavage at pH 5.5 or below. However, upon exposure to neutral pH following VP2 cleavage, its VP1-specific sequences and genome are extruded even at room temperature, underscoring the significance of the VP2 cleavage step for MVM particle dynamics.

Parvoviral virions deploy a capsid-tethered lipolytic enzyme to breach the endosomal membrane during cell entry.

Enveloped viruses deliver their virions into the host cell by fusion with the cellular plasma or endosomal membrane, thus creating topological continuity between the cytosol and the inside of the viral envelope. Nonenveloped viruses are, by their very nature, denied this strategy and must employ alternative methods to breach their host cell's delimiting membrane. We show here that the compact icosahedral parvoviral virion gains entry by deploying a lipolytic enzyme, phospholipase A(2) (PLA(2)), that is expressed at the N terminus of VP1, the minor coat protein. This region of VP1 is normally sequestered within the viral shell but is extruded during the entry process as a capsid-tethered domain. A single amino acid substitution in the active site of the VP1 PLA(2) inactivates enzymatic activity and abrogates infectivity. We have used transencapsidation of a vector expressing green fluorescent protein to show that infection by this PLA(2)-defective mutant can be complemented by coinfection with wild-type or mutant full virions, provided they can express a functional PLA(2). Even though wild-type empty capsids contain an active form of the enzyme, it is not externalized under physiological conditions, and such capsids are not able to complement the PLA(2) mutant. Significantly, highly efficient rescue can be achieved by polyethyleneimine-induced endosome rupture or by coinfection with adenovirus as long as uptake of the two viruses is simultaneous and the adenovirus is capable of deploying pVI, a capsid protein with endosomolytic activity. Together, these results demonstrate a previously unrecognized enzymatic mechanism for nonenveloped virus penetration.

A conserved leucine that constricts the pore through the capsid fivefold cylinder plays a central role in parvoviral infection.

The atomic structure of the DNA-containing T = 1 particle of the parvovirus minute virus of mice (MVM) reveals cylindrical projections at each fivefold symmetry axis, each containing an 8 Angstrom pore through which runs 10 amino acids of a single VP2 N-terminus. The tightest constriction of this pore is formed at its inner end by the juxtaposition of leucine side chains from position 172 of five independent VP2 molecules. To test whether L172 modulates the extrusion of VP N-termini, we constructed and analyzed a complete set of amino acid substitution mutants at this highly conserved residue. All but one mutant produced DNA-containing virions, but only two, L172V and L172I, were infectious, the others being blocked for viral entry. Several mutants were significantly defective for assembly at 39 degrees C, but not at 32 degrees C. L172W significantly impaired genome encapsidation, indicating that the fivefold cylinder may also be the DNA packaging portal. Although tryptic cleavage of the VP2 N-terminus was not affected for the mutants, VP1 was degraded during proteolysis of mutant, but not wild-type, virions.