Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome activity.

The immunoproteasome (iCP) can be expressed under inflammatory conditions, such as exposure to interferon-gamma (IFN-γ), that alerts the cell to begin generating iCP preferentially over the standard proteasome (sCP). With the iCP becoming a widely targeted isoform in a variety of diseases, there is a need to understand its activity and expression in cells and in vivo. Activity-based probes for the iCP have been developed but their application has been limited due to their difficult synthesis and cannot be used in tissues or whole animals. Our lab has previously demonstrated we can monitor iCP activity using a 4-mer peptide linked to a fluorophore and a peptoid. This was utilized in the development of the first cell-permeable iCP activity-based probe that did not include a covalent reactive moiety. Here, we demonstrate that this same peptide recognition sequence can be appended to aminoluciferin, caging it, until its interaction with the iCP. This probe should be applicable to monitor iCP activity in animal models where tumor or other tissue has been engineered to produce luciferase. We anticipate it could also be applied to observe iCP activity as tumors are formed in vivo.

The transition from weak (noncovalent) interactions to fully developed covalent bonds is examined using the quantum theory of atoms in molecules in a series of halogen-bonded (XB) complexes of bromosubstituted electrophiles, RBr, with 1,4-diazabicyclo[2.2.2]octane (DABCO) and Cl- and Br- anions. The gradual decrease in the XB lengths in these associations, d Br···Y (where Y = Cl-, Br-, or N), was accompanied by the exponential increase in the binding energies and charge transfer, as well as electron densities and magnitudes of the kinetic and potential energy densities at the bond critical points (BCPs) on the Br···Y bond path. These indices, as well as characteristics of the adjacent bonds in the XB donor, followed remarkably close trend lines when plotted against the normalized XB length R BrY = d Br···Y/(r Br + r Y) (where r Br and r Y are the van der Waals radii) regardless of the methods [MP2/6-311+G(d,p) or M062X/6-311+G(d,p)], media (gas phase or dichloromethane), and nucleophiles (Cl-, Br-, or DABCO). In the systems with an R BrY higher than about 0.78, the energy densities H(r) at BCPs at the Br···Y bond path were small and positive, and XBs did not substantially affect the characteristics of the adjacent R-Br covalent bond in the XB donor. Accordingly, the XB can be identified as noncovalent in this range. In the complexes with R BrY values between about 0.67 and 0.78, energy densities H(r) at Br···Y BCPs were negative, and their magnitudes increased with the decrease in the Br···Y separation. In this range, formation of XBs was accompanied by the increase in the R-Br bond length in the XB donor and the decrease in the magnitude of the (negative) H(r) values at the BCPs of the R-Br bonds. XBs can be classified as partially covalent in this R BrY range. At an R BrY less than about 0.67, electron densities were larger, and energy densities were more negative at BCPs of the Br···Y bond than those at BCPs of the R-Br bond in the XB donor. This indicates that Br···Y bonds were stronger than R-Br bonds, and these (Br···Y) XBs can be regarded as essentially covalent. The synchronous change of a variety of (R-Br and Br···Y) bonding characteristics with R BrY suggests that the normalized XB bond length can be used as a basic parameter in the identification of the type of intermolecular interaction. A continuity of these characteristics suggests an inherent relationship between limiting (covalent and noncovalent) types of XBs and thus an onset of molecular-orbital interactions in the weaker bonds.

Halogen-bonded (XB) complexes between halide anions and a cyclopropenylium-based anionic XB donor were characterized in solution for the first time. Spontaneous formation of such complexes confirms that halogen bonding is sufficiently strong to overcome electrostatic repulsion between two anions. The formation constants of such anti-electrostatic associations are comparable to those formed by halides with neutral halogenated electrophiles. However, while the latter usually show charge-transfer absorption bands, the UV-Vis spectra of the anion-anion complexes examined herein are determined by the electronic excitations within the XB donor. The identification of XB anion-anion complexes substantially extends the range of the feasible XB systems, and it provides vital information for the discussion of the nature of this interaction.

The ubiquitin-proteasome system serves as the major proteolytic degradation pathway in eukaryotic cells. Many inhibitors that covalently bind to the proteasomes active sites have been developed for hematological cancers, but resistance can arise in patients. To overcome limitations of active-site proteasome inhibitors, we and others have focused on developing ligands that target subunits on the 19S regulatory particle (19S RP). One such 19S RP subunit, Rpn-13, is a ubiquitin receptor required for hematological cancers to rapidly degrade proteins to avoid apoptosis. Reported Rpn-13 inhibitors covalently bind to the Rpn-13s Pru domain and have been effective anti-hematological cancer agents. Here, we describe the discovery of TCL-1, a non-covalent binder to the Pru domain. Optimization of TCL-1s carboxylate group to an ester increases its cytotoxicity in hematological cancer cell lines. Altogether, our data provides a new scaffold for future medicinal chemistry optimization to target Rpn-13 therapeutically.