γ-Secretase inhibitors (GSIs) and modulators (GSMs) are at the frontline of cancer and Alzheimer's disease research, respectively. While both are therapeutically promising, not much is known about their interactions with proteins other than γ-secretase. Signal peptide peptidase (SPP), like γ-secretase, is a multispan transmembrane aspartyl protease that catalyzes regulated intramembrane proteolysis. We used active site-directed photophore walking probes to study the effects of different GSIs and GSMs on the active sites of γ-secretase and SPP and found that nontransition state GSIs inhibit labeling of γ-secretase by activity-based probes but enhance labeling of SPP. The opposite is true of GSMs, which have little effect on the labeling of γ-secretase but diminish labeling of SPP. These results demonstrate that GSIs and GSMs are altering the structure of not only γ-secretase but also SPP, leading to potential changes in enzyme activity and specificity that may impact the clinical outcomes of these molecules.
SPRY domain- and SOCS box-containing proteins SPSB1, SPSB2, and SPSB4 interact with inducible nitric oxide synthase (iNOS), causing the iNOS to be polyubiquitinated and targeted for degradation. Inhibition of this interaction increases iNOS levels, and consequently cellular nitric oxide (NO) concentrations, and has been proposed as a potential strategy for killing intracellular pathogens. We previously described two DINNN-containing cyclic peptides (CP1 and CP2) as potent inhibitors of the murine SPSB-iNOS interaction. In this study, we report the crystal structures of human SPSB4 bound to CP1 and CP2 and human SPSB2 bound to CP2. We then used these structures to design a new inhibitor in which an intramolecular hydrogen bond was replaced with a hydrocarbon linkage to form a smaller macrocycle while maintaining the bound geometry of CP2 observed in the crystal structures. This resulting pentapeptide SPSB-iNOS inhibitor (CP3) has a reduced macrocycle ring size, fewer nonbinding residues, and includes additional conformational constraints. CP3 has a greater affinity for SBSB2 ( KD = 7 nM as determined by surface plasmon resonance) and strongly inhibits the SPSB2-iNOS interaction in macrophage cell lysates. We have also determined the crystal structure of CP3 in complex with human SPSB2, which reveals the structural basis for the increased potency of CP3 and validates the original design.
There is a need to improve and extend the use of clinically approved poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi), including for BRCA wild-type triple-negative breast cancer (TNBC). The demonstration that ruthenium(II) polypyridyl complex (RPC) metallointercalators can rapidly stall DNA replication fork progression provides the rationale for their combination alongside DNA damage response (DDR) inhibitors to achieve synergism in cancer cells. The aim of the present study was to evaluate use of the multi-intercalator [Ru(dppz)2(PIP)]2+ (dppz = dipyrido[3,2-a:2',3'-c]phenazine, PIP = (2-(phenyl)imidazo[4,5-f][1,10]phenanthroline, Ru-PIP) alongside the PARPi olaparib and NU1025. Cell proliferation and clonogenic survival assays indicated a synergistic relationship between Ru-PIP and olaparib in MDA-MB-231 TNBC and MCF7 human breast cancer cells. Strikingly, low dose Ru-PIP renders both cell lines hypersensitive to olaparib, with a >300-fold increase in olaparib potency in TNBC, the largest nongenetic PARPi enhancement effect described to date. A negligible impact on the viability of normal human fibroblasts was observed for any combination tested. Increased levels of DNA double-strand break (DSB) damage and olaparib abrogation of Ru-PIP-activated pChk1 signaling are consistent with PARPi-facilitated collapse of Ru-PIP-associated stalled replication forks. This results in enhanced G2/M cell-cycle arrest, apoptosis, and decreased cell motility for the combination treatment compared to single-agent conditions. This work establishes that an RPC metallointercalator can be combined with PARPi for potent synergy in BRCA-proficient breast cancer cells, including TNBC.