Membrane targeting with palmitoylated lysine added to PP1-disrupting peptide induces PP1-independent signaling.

Protein phosphatase-1 (PP1) is a ubiquitous enzyme involved in multiple processes inside cells. PP1-disrupting peptides (PDPs) are chemical tools that selectively bind to PP1 and release its activity. To restrict the activity of PDPs to a cellular compartment, we developed PDP-Mem, a cell membrane-targeting PDP. The membrane localization was achieved through the introduction of a palmitoylated lysine. PDP-Mem was shown to activate PP1α in vitro and to localize to the membrane of HeLa Kyoto and U2OS cells. However, in cells, the combination of the polybasic sequence for cell penetration and the membrane targeting palmitoylated lysine activates the MAPK signaling pathway and induces cytoplasmic calcium release independently of PP1 activation. Therefore, when targeting peptides to cellular membranes, undesired effects induced by the targeting sequence and lipid modification need to be considered.

PLDMS: Phosphopeptide Library Dephosphorylation Followed by Mass Spectrometry Analysis to Determine the Specificity of Phosphatases for Dephosphorylation Site Sequences.

A detailed understanding of the sequence preference surrounding phosphorylation sites is essential for deciphering the function of the human phosphoproteome . Whereas the mechanisms for substrate site recognition by kinases are relatively well understood, the selection mechanisms for the corresponding phosphatases pose several obstacles. However, multiple pieces of evidence point towards a role of the amino acid sequence in the direct vicinity of the phosphorylation site for recognition by phosphatase enzymes. Peptide library-based studies for enzymes attaching posttranslational modifications (PTMs) are relatively straight forward to carry out. However, studying enzymes removing PTMs pose a challenge in that libraries with a PTM attached are needed as a starting point. Here, we present our methodology using large synthetic phosphopeptide libraries to study the preferred sequence context of protein phosphatases. The approach, termed "phosphopeptide library dephosphorylation followed by mass spectrometry" (PLDMS), allows for the exact control of phosphorylation site incorporation and the synthetic route is capable of covering several thousand peptides in a single tube reaction. Furthermore, it enables the user to analyze MS data tailored to the needs of a specific library and thereby increase data quality. We therefore expect a wide applicability of this technique for a range of enzymes catalyzing the removal of PTMs.

Dissecting the sequence determinants for dephosphorylation by the catalytic subunits of phosphatases PP1 and PP2A.

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and phosphothreonine (pThr), and are involved in virtually all cellular processes and numerous diseases. The catalytic subunits exist in cells in form of holoenzymes, which impart substrate specificity. The contribution of the catalytic subunits to the recognition of substrates is unclear. By developing a phosphopeptide library approach and a phosphoproteomic assay, we demonstrate that the specificity of PP1 and PP2A holoenzymes towards pThr and of PP1 for basic motifs adjacent to the phosphorylation site are due to intrinsic properties of the catalytic subunits. Thus, we dissect this amino acid specificity of the catalytic subunits from the contribution of regulatory proteins. Furthermore, our approach enables discovering a role for PP1 as regulator of the GRB-associated-binding protein 2 (GAB2)/14-3-3 complex. Beyond this, we expect that this approach is broadly applicable to detect enzyme-substrate recognition preferences.

Structural and mechanistic insights into the interaction of the circadian transcription factor BMAL1 with the KIX domain of the CREB-binding protein.

The mammalian CLOCK:BMAL1 transcription factor complex and its coactivators CREB-binding protein (CBP)/p300 and mixed-lineage leukemia 1 (MLL1) critically regulate circadian transcription and chromatin modification. Circadian oscillations are regulated by interactions of BMAL1's C-terminal transactivation domain (TAD) with the KIX domain of CBP/p300 (activating) and with the clock protein CRY1 (repressing) as well as by the BMAL1 G-region preceding the TAD. Circadian acetylation of Lys537 within the G-region enhances repressive BMAL1-TAD-CRY1 interactions. Here, we characterized the interaction of the CBP-KIX domain with BMAL1 proteins, including the BMAL1-TAD, parts of the G-region, and Lys537 Tethering the small compound 1-10 in the MLL-binding pocket of the CBP-KIX domain weakened BMAL1 binding, and MLL1-bound KIX did not form a ternary complex with BMAL1, indicating that the MLL-binding pocket is important for KIX-BMAL1 interactions. Small-angle X-ray scattering (SAXS) models of BMAL1 and BMAL1:KIX complexes revealed that the N-terminal BMAL1 G-region including Lys537 forms elongated extensions emerging from the bulkier BMAL1-TAD:KIX core complex. Fitting high-resolution KIX domain structures into the SAXS-derived envelopes suggested that the G-region emerges near the MLL-binding pocket, further supporting a role of this pocket in BMAL1 binding. Additionally, mutations in the second CREB-pKID/c-Myb-binding pocket of the KIX domain moderately impacted BMAL1 binding. The BMAL1(K537Q) mutation mimicking Lys537 acetylation, however, did not affect the KIX-binding affinity, in contrast to its enhancing effect on CRY1 binding. Our results significantly advance the mechanistic understanding of the protein interaction networks controlling CLOCK:BMAL1- and CBP-dependent gene regulation in the mammalian circadian clock.

NLRP3 Inflammasome Inhibitor Ameliorates Amyloid Pathology in a Mouse Model of Alzheimer's Disease.

The activation of the NLRP3 inflammasome signaling pathway plays an important role in the neuroinflammation in Alzheimer's disease (AD). In this study, we investigated the effects of JC-124, a rationally designed NLRP3 inflammasome inhibitor, on AD-related deficits in CRND8 APP transgenic mice (TgCRND8). We first demonstrated increased formation and activation of NLRP3 inflammasome in TgCRND8 mice compared to non-transgenic littermate controls, which was inhibited by the treatment with JC-124. Importantly, JC-124 treatment led to decreased levels of Aß deposition and decreased levels of soluble and insoluble Aß1-42 in the brain of CRND8 mice which was accompanied by reduced ß-cleavage of APP, reduced activation of microglia but enhanced astrocytosis. Oxidative stress was decreased and synaptophysin was increased in the CRND8 mice after JC-124 treatment, demonstrating a neuroprotective effect. Overall, these data demonstrated beneficial effects of JC-124 as a specific NLRP3 inflammasome inhibitor in AD mouse model and supported the further development of NLRP3 inflammasome inhibitors as a viable option for AD therapeutics.

Development and Characterization of a Hydroxyl-Sulfonamide Analogue, 5-Chloro-N-[2-(4-hydroxysulfamoyl-phenyl)-ethyl]-2-methoxy-benzamide, as a Novel NLRP3 Inflammasome Inhibitor for Potential Treatment of Multiple Sclerosis.

In our efforts to develop novel small-molecule inhibitors for the NOD-like receptor family pyrin-domain-containing 3 (NLRP3) inflammasome as potential disease-modifying agents to treat neurological disorders including multiple sclerosis (MS), a hydroxyl sulfonamide analogue JC-171 has been rationally designed and biologically characterized both in vitro and in vivo. Our studies established that JC-171 dose dependently inhibited LPS/ATP-induced interleukin-1ß (IL-1ß) release from J774A.1 macrophages with an IC50 of 8.45 ± 1.56 µM. Selective inhibition of the NLRP3 inflammasome induced IL-1ß release by this compound was also confirmed using mouse bone-marrow-derived macrophages and LPS-challenged mice in vivo. Furthermore, immunoprecipitation study revealed that JC-171 interfered with NLRP3/ASC interaction induced by LPS/ATP stimulation. More importantly, JC-171 treatment delayed the progression and reduced the severity of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, in both prophylactic and therapeutic settings. This coincided with blocking of IL-1ß production and a pathogenic Th17 response. Collectively, these results suggest that JC-171 is a selective NLRP3 inflammasome inhibitor with biological activity in vivo, thus strongly encouraging further development of this lead compound as a potential therapeutic agent for human MS.

Mechanistic Insight of Bivalent Compound 21MO as Potential Neuroprotectant for Alzheimer's Disease.

We have recently developed a bivalent strategy to provide novel compounds that potentially target multiple risk factors involved in the development of Alzheimer's disease (AD). Our previous studies employing a bivalent compound with a shorter spacer (17MN) implicated that this compound can localize into mitochondria and endoplasmic reticulum (ER), thus interfering with the change of mitochondria membrane potential (MMP) and Ca(2+) levels in MC65 cells upon removal of tetracycline (TC). In this report, we examined the effects by a bivalent compound with a longer spacer (21MO) in MC65 cells. Our results demonstrated that 21MO suppressed the change of MMP, possibly via interaction with the mitochondrial complex I in MC65 cells. Interestingly, 21MO did not show any effects on the Ca(2+) level upon TC removal in MC65 cells. Our previous studies suggested that the mobilization of Ca(2+) in MC65 cells, upon withdraw of TC, originated from ER, so the results implicated that 21MO may preferentially interact with mitochondria in MC65 cells under the current experimental conditions. Collectively, the results suggest that bivalent compounds with varied spacer length and cell membrane anchor moiety may exhibit neuroprotective activities via different mechanisms of action.

Bivalent ligands incorporating curcumin and diosgenin as multifunctional compounds against Alzheimer's disease.

In an effort to combat the multifaceted nature of Alzheimer's disease (AD) progression, a series of multifunctional, bivalent compounds containing curcumin and diosgenin were designed, synthesized, and biologically characterized. Screening results in MC65 neuroblastoma cells established that compound 38 with a spacer length of 17 atoms exhibited the highest protective potency with an EC50 of 111.7 ± 9.0 nM. A reduction in protective activity was observed as spacer length was increased up to 28 atoms and there is a clear structural preference for attachment to the methylene carbon between the two carbonyl moieties of curcumin. Further study suggested that antioxidative ability and inhibitory effects on amyloid-ß oligomer (AßO) formation may contribute to the neuroprotective outcomes. Additionally, compound 38 was found to bind directly to Aß, similar to curcumin, but did not form complexes with the common biometals Cu, Fe, and Zn. Altogether, these results give strong evidence to support the bivalent design strategy in developing novel compounds with multifunctional ability for the treatment of AD.

Bivalent Compound 17MN Exerts Neuroprotection through Interaction at Multiple Sites in a Cellular Model of Alzheimer's Disease.

Multiple pathogenic factors have been suggested to play a role in the development of Alzheimer's disease (AD). The multifactorial nature of AD also suggests the potential use of compounds with polypharmacology as effective disease-modifying agents. Recently, we have developed a bivalent strategy to include cell membrane anchorage into the molecular design. Our results demonstrated that the bivalent compounds exhibited multifunctional properties and potent neuroprotection in a cellular AD model. Herein, we report the mechanistic exploration of one of the representative bivalent compounds, 17MN, in MC65 cells. Our results established that MC65 cells die through a necroptotic mechanism upon the removal of tetracycline (TC). Furthermore, we have shown that mitochondrial membrane potential and cytosolic Ca2+ levels are increased upon removal of TC. Our bivalent compound 17MN can reverse such changes and protect MC65 cells from TC removal induced cytotoxicity. The results also suggest that 17MN may function between the Aß species and RIPK1 in producing its neuroprotection. Colocalization studies employing a fluorescent analog of 17MN and confocal microscopy demonstrated the interactions of 17MN with both mitochondria and endoplasmic reticulum, thus suggesting that 17MN exerts its neuroprotection via a multiple-site mechanism in MC65 cells. Collectively, these results strongly support our original design rationale of bivalent compounds and encourage further optimization of this bivalent strategy to develop more potent analogs as novel disease-modifying agents for AD.

Curcumin/melatonin hybrid 5-(4-hydroxy-phenyl)-3-oxo-pentanoic acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide ameliorates AD-like pathology in the APP/PS1 mouse model.

In our efforts to develop hybrid compounds of curcumin and melatonin as potential disease-modifying agents for Alzheimer's disease (AD), a potent lead hybrid compound, Z-CM-I-1, has been recently identified and biologically characterized in vitro. In this work, we report the in vivo effects of Z-CM-I-1 on AD pathologies in an APP/PS1 transgenic AD model. Our studies demonstrated that Z-CM-I-1 significantly decreased the accumulation of Aß in the hippocampus and cortex regions of the brain and reduced inflammatory responses and oxidative stress after treatment for 12 weeks at 50 mg/kg per dose via oral administration. Furthermore, Z-CM-I-1 significantly improved synaptic dysfunction evidenced by the increased expression of synaptic marker proteins, PSD95 and synaptophysin, indicating its protective effects on synaptic degeneration. Lastly, we demonstrated that Z-CM-I-1 significantly increased the expression level of complexes I, II, and IV of the mitochondria electron transport chain in the brain tissue of APP/PS1 mice. Collectively, these results clearly suggest that Z-CM-I-1 is orally available and exhibits multifunctional properties in vivo on AD pathologies, thus strongly encouraging further development of this lead compound as a potential disease-modifying agent for AD patients.

Discovery of 5-(4-hydroxyphenyl)-3-oxo-pentanoic acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide as a neuroprotectant for Alzheimer's disease by hybridization of curcumin and melatonin.

In our effort to develop effective neuroprotectants as potential treatments for Alzheimer's disease (AD), hybrid compounds of curcumin and melatonin, two natural products that have been extensively studied in various AD models, were designed, synthesized, and biologically characterized. A lead hybrid compound (7) was discovered to show significant neuroprotection with nanomolar potency (EC50 = 27.60 ± 9.4 nM) in MC65 cells, a cellular AD model. Multiple in vitro assay results established that 7 exhibited moderate inhibitory effects on the production of amyloid-ß oligomers (AßOs) in MC65 cells, but not on the aggregation of Aß species. It also exhibited significant antioxidative properties. Further mechanistic studies demonstrated that 7's antioxidant effects correlate well with its neuroprotective potency for MC65 cells, and these effects might be due to its interference with the interactions of AßOs within the mitochondria of MC65 cells. Furthermore, 7 was confirmed to cross the blood-brain barrier (BBB) and deliver a sufficient amount to brain tissue after oral administration. Collectively, these results strongly support the hybridization approach as an efficient strategy to help identify novel scaffolds with a desired pharmacology, and strongly encourage further optimization of 7 to develop more potent neuroprotectants for AD.