The endogenous opioid pentapeptides [Met5]enkephalin (H-TyrGly-Gly-Phe-Met-OH) and [Leu5]enkephalin (H-Tyr-Gly-GlyPhe-Leu-OH) have been shown to interact with several classes of opioid receptors (1-3) that may mediate different physiological responses. Elucidation of the roles of the individual receptor classes has been hampered by the general lack of enkephalin analogs with a high degree of selectivity for a single receptor type. The vast majority of analogs crossreact extensively with the different receptors, making it difficult to define receptor roles. This situation has been in part ameliorated by recent reports of an enkephalin analog highly selective for the ,At opioid receptor (4-6) and a nonpeptide opiate with high K receptor selectivity (7). However, analogs with corresponding selectivity for the 8 opioid receptor have not been demonstrated.One approach for the design of more selective analogs involves the incorporation of conformational restrictions. The native enkephalins, like most small, linear peptides, possess considerable conformational flexibility and by virtue of this flexibility can attain the presumably different conformational features required for interaction with different classes of opioid receptors. In principle, appropriate restriction of this flexibility can lead to analogs able to assume the conformation required to interact favorably with only one class of receptor. One method for effecting conformational restrictions is via cyclization of the peptide that constrains the resulting analog to assume a compact topography. Several active, cyclic enkephalin analogs have been reported, all of which are cyclized by either side chain to carboxyl terminus (8,9) It has previously been shown that, in aqueous solution, the tocin ring portion of Pen-containing oxytocin analogs is conformationally restricted, whereas the tocin ring of oxytocin itself is quite flexible (13-16). This difference arises from the rigidifying effect of gem-dialkyl substituents in medium-sized rings and suggests that the 8 (Fig. 1) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (K = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.

No abstract

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins.

A detailed examination is made of several approximations to the first-order Hartree—Fock perturbation equation. Four distinct methods are considered: the coupled (Method a) and the uncoupled (Method c) approximation of Dalgarno, a new alternative uncoupled approximation (Method b), and the simplified uncoupled approximation of Karplus and Kolker (Method d). By a comparison of the pertinent equations, it is shown that Methods a, b, and d correspond to each other in the use of a core potential analogous to that appearing in the zeroth-order Hartree—Fock equation, while Method c differs due to the inclusion of an extraneous self-potential term. An alternative analysis based on an orbital basis set expansion of the perturbed function demonstrates that Method c has an energy denominator of the simple Hückel type, while both Methods a and b include important two-electron correction terms. Also, it is found that of the four approximations, only Method c can be obtained as the first-order correction with a zeroth-order many-electron Hamiltonian that has the Hartree—Fock determinant as its eigenfunction; the other techniques are one electron in character, as is the Hartree—Fock method itself. The significance of the difference in the core potential is demonstrated by test calculations of dipole and quadrupole polarizabilities and shielding factors for the two-, three-, and four-electron isoelectronic series. A variational technique is used with a trial function that has the form of a polynomial times the unperturbed orbital. For the polarizabilities, it is found that Method b is an excellent approximation to Method a, indicating that the self-consistency condition on the Method a solutions has a very small effect. The shielding factors, however, appear to be more sensitive to the self-consistency requirement. Both Method c and Method d introduce larger errors than Method b, with Method c particularly poor for three-electron atoms and ions. The constraint introduced by choice of trial function is shown to be unimportant for polarizabilities, but quite severe for the four-electron atom shielding factors. A comparison of the complexity of the various techniques shows that the relative computing times for Methods a, b, c, and d are in the ratio of 300 to 60 to 75 to 1. Thus, Method d is simplest by far, although its speed is achieved by some loss of accuracy with respect to Methods a and b.

Phenotypic screening of compound libraries is a significant trend in drug discovery, yet success can be hindered by difficulties in identifying the underlying cellular targets. Current approaches rely on tethering bioactive compounds to a capture tag or surface to allow selective enrichment of interacting proteins for subsequent identification by mass spectrometry. Such methods are often constrained by ineffective capture of low affinity and low abundance targets. In addition, these methods are often not compatible with living cells and therefore cannot be used to verify the pharmacological activity of the tethered compounds. We have developed a novel chloroalkane capture tag that minimally affects compound potency in cultured cells, allowing binding interactions with the targets to occur under conditions relevant to the desired cellular phenotype. Subsequent isolation of the interacting targets is achieved through rapid lysis and capture onto immobilized HaloTag protein. Exchanging the chloroalkane tag for a fluorophore, the putative targets identified by mass spectrometry can be verified for direct binding to the compound through resonance energy transfer. Using the interaction between histone deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model system, we were able to identify and verify all the known HDAC targets of SAHA as well as two previously undescribed targets, ADO and CPPED1. The discovery of ADO as a target may provide mechanistic insight into a reported connection between SAHA and Huntington's disease.

No abstract

G protein–coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the β-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.