Are Hemorrhagic Cysts Hyperintense Enough on T1-Weighted MRI to Be Distinguished From Renal Cell Carcinomas? A Retrospective Analysis of 204 Patients.

OBJECTIVE. The purpose of this study was to evaluate the utility of T1- and T2-weighted MRI signal-intensity ratios and signal-intensity SDs of renal lesions to determine the feasibility of distinguishing between simple cysts, hemorrhagic renal cysts, clear cell renal cell carcinoma (RCC), and papillary RCC. MATERIALS AND METHODS. Pathology records of 53 cases of papillary RCCs between 1 and 5 cm in size were included. Thirty-eight pathology-proven clear cell RCCs, 54 simple renal cysts seen on abdominal MRI, and 59 hemorrhagic renal cysts seen on abdominal MRI were identified. Lesion location and size, T1- and T2-weighted signal intensity, and corresponding SD values for each renal lesion and psoas muscle (from which lesion-to-muscle ratios were calculated) were collected. RESULTS. Analysis revealed a statistically significant difference (p < 0.001) in T1-weighted lesion-to-muscle signal-intensity ratios between simple cysts (mean ± standard error, 0.54 ± 0.05), clear cell RCCs (0.86 ± 0.06), papillary RCCs (1.17 ± 0.05), and hemorrhagic renal cysts (1.95 ± 0.04). The T2-weighted lesion-to-muscle signal-intensity ratios showed a statistically significant difference between all lesion types (p < 0.02) except between hemorrhagic renal cysts and papillary RCCs, where the difference approached significance (p = 0.075). ROC analysis showed an optimal cutoff of T1-weighted lesion-to-muscle signal-intensity ratio of 1.39 to differentiate hemorrhagic cysts (above this value) from RCCs (below this value). Corresponding sensitivity and specificity were 91.2% and 74.6%, respectively. CONCLUSION. T1-weighted lesion-to-muscle signal-intensity ratio is a useful measure to discriminate mildly hyperintense RCCs from more hyperintense hemorrhagic cysts when contrast enhancement is unavailable.

CoREST Complex-Selective Histone Deacetylase Inhibitors Show Prosynaptic Effects and an Improved Safety Profile To Enable Treatment of Synaptopathies.

Synaptic dysfunction is a pathological feature in many neurodegenerative disorders, including Alzheimer's disease, and synaptic loss correlates closely with cognitive decline. Histone deacetylases (HDACs) are involved in chromatin remodeling and gene expression and have been shown to regulate synaptogenesis and synaptic plasticity, thus providing an attractive drug discovery target for promoting synaptic growth and function. To date, HDAC inhibitor compounds with prosynaptic effects are plagued by known HDAC dose-limiting hematological toxicities, precluding their application to treating chronic neurologic conditions. We have identified a series of novel HDAC inhibitor compounds that selectively inhibit the HDAC-co-repressor of repressor element-1 silencing transcription factor (CoREST) complex while minimizing hematological side effects. HDAC1 and HDAC2 associate with multiple co-repressor complexes including CoREST, which regulates neuronal gene expression. We show that selectively targeting the CoREST co-repressor complex with the representative compound Rodin-A results in increased spine density and synaptic proteins, and improved long-term potentiation in a mouse model at doses that provide a substantial safety margin that would enable chronic treatment. The CoREST-selective HDAC inhibitor Rodin-A thus represents a promising therapeutic strategy in targeting synaptic pathology involved in neurologic disorders.

Investigating small molecules to inhibit germinal center kinase-like kinase (GLK/MAP4K3) upstream of PKCθ phosphorylation: Potential therapy to modulate T cell dependent immunity.

Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines.

Germinal-center kinase-like kinase co-crystal structure reveals a swapped activation loop and C-terminal extension.

Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.

ATP-Competitive MLKL Binders Have No Functional Impact on Necroptosis.

MLKL is a pore forming pseudokinase involved in the final stage of necroptosis, a form of programmed cell death. Its phosphorylation by RIPK3 is necessary for triggering necroptosis but not for triggering apoptosis, which makes it a unique target for pharmacological inhibition to block necroptotic cell death. This mechanism has been described as playing a role in disease progression in neurodegenerative and inflammatory diseases. A type II kinase inhibitor (cpd 1) has been described that reportedly binds to the MLKL pseudokinase domain and prevents necroptosis. Here we describe five compounds that bind to the MLKL ATP-binding site, however the four MLKL-selective compounds have no activity in rescuing cells from necroptosis. We use kinase selectivity panels, crystallography and a new conformationally sensitive method of measuring protein conformational changes (SHG) to confirm that the one previously reported compound that can rescue cells (cpd 1) is a non-selective type II inhibitor that also inhibits the upstream kinase RIPK1. Although this compound can shift the GFE motif of the activation loop to an "out" position, the accessibility of the key residue Ser358 in the MLKL activation loop is not affected by compound binding to the MLKL active site. Our studies indicate that an ATP-pocket inhibitor of the MLKL pseudokinase domain does not have any impact on the necroptosis pathway, which is contrary to a previously reported study.

Minimization of drug-drug interaction risk and candidate selection in a natural product-based class of gamma-secretase modulators.

Early lead compounds in this gamma secretase modulator series were found to potently inhibit CYP3A4 and other human CYP isoforms increasing their risk of causing drug-drug-interactions (DDIs). Using structure-activity relationships and CYP3A4 structural information, analogs were developed that minimized this DDI potential. Three of these new analogs were further characterized by rat PK, rat PK/PD and rat exploratory toxicity studies resulting in selection of SPI-1865 (14) as a preclinical development candidate.

Natural Product and Natural Product-Derived Gamma Secretase Modulators from Actaea Racemosa Extracts.

Alzheimer's disease is characterized by pathogenic oligomerization, aggregation, and deposition of amyloid beta peptide (Aß), resulting in severe neuronal toxicity and associated cognitive dysfunction. In particular, increases in the absolute or relative level of the major long form of Aß, Aß42, are associated with increased cellular toxicity and rapidity of disease progression. As a result of this observation, screening to identify potential drugs to reduce the level of Aß42 have been undertaken by way of modulating the proteolytic activity of the gamma secretase complex without compromising its action on other essential substrates such as Notch. In this review we summarize results from a program that sought to develop such gamma secretase modulators based on novel natural products identified in the extract of Actaea racemosa, the well-known botanical black cohosh. Following isolation of compound 1 (SPI-014), an extensive medicinal chemistry effort was undertaken to define the SAR of 1 and related semisynthetic compounds. Major metabolic and physicochemical liabilities in 1 were overcome including replacement of both the sugar and acetate moieties with more stable alternatives that improved drug-like properties and resulted in development candidate 25 (SPI-1865). Unanticipated off-target adrenal toxicity, however, precluded advancement of this series of compounds into clinical development.

Efficacy of SPI-1865, a novel gamma-secretase modulator, in multiple rodent models.

INTRODUCTION: Modulation of the gamma-secretase enzyme, which reduces the production of the amyloidogenic Aß42 peptide while sparing the production of other Aß species, is a promising therapeutic approach for the treatment of Alzheimer's disease. Satori has identified a unique class of small molecule gamma-secretase modulators (GSMs) capable of decreasing Aß42 levels in cellular and rodent model systems. The compound class exhibits potency in the nM range in vitro and is selective for lowering Aß42 and Aß38 while sparing Aß40 and total Aß levels. In vivo, a compound from the series, SPI-1865, demonstrates similar pharmacology in wild-type CD1 mice, Tg2576 mice and Sprague Dawley rats. METHODS: Animals were orally administered either a single dose of SPI-1865 or dosed for multiple days. Aß levels were measured using a sensitive plate-based ELISA system (MSD) and brain and plasma exposure of drug were assessed by LC/MS/MS. RESULTS: In wild-type mice using either dosing regimen, brain Aß42 and Aß38 levels were decreased upon treatment with SPI-1865 and little to no statistically meaningful effect on Aß40 was observed, reflecting the changes observed in vitro. In rats, brain Aß levels were examined and similar to the mouse studies, brain Aß42 and Aß38 were lowered. Comparable changes were also observed in the Tg2576 mice, where Aß levels were measured in brain as well as plasma and CSF. CONCLUSIONS: Taken together, these data indicate that SPI-1865 is orally bioavailable, brain penetrant, and effective at lowering Aß42 in a dose responsive manner. With this unique profile, the class of compounds represented by SPI-1865 may be a promising new therapy for Alzheimer's disease.

An improved cell-based method for determining the γ-secretase enzyme activity against both Notch and APP substrates.

γ-Secretase modulators (GSM), which reduce amyloidogenic Aß(42) production while maintaining total Aß levels, and Notch-sparing γ-secretase inhibitors (GSIs) are promising therapies for the treatment of Alzheimer's Disease (AD). To have a safety margin for therapeutic use, GSMs and GSIs need to allow Notch intracellular domain (NICD) production, while preventing neurotoxic Aß peptide production. Typically, GSI and GSM effects on these substrates are determined using two different cell lines, one for the measurement of enzyme activity against each substrate. However, predicting selectivity for different substrates across cell systems may reduce the reliability of such ratios such that the in vitro data are not useful for predicting in vivo safety margins. This is especially concerning since the IC(50)'s of some GSIs vary depending upon the level of APP expression in a cell line. To circumvent this problem, we utilized the SUP-T1 cell line which expresses a truncated Notch receptor fragment that does not need sheddase cleavage to be a γ-secretase substrate. When combined with a sensitive method of measuring Aß production, this assay system allows both substrates to be measured simultaneously, reducing the potential to calculate imprecise selectivity margins. To demonstrate the value of this system, known GSIs and GSMs were examined in the SUP-T1 dual substrate assay. IC(50)'s were determined for both substrates and the in vitro selectivity margin was calculated. These data suggest using a single cell line is a more accurate prediction of the fold difference between NICD inhibition and Aß(42) lowering for therapeutically promising GSIs and GSMs.

Discovery of a novel pharmacological and structural class of gamma secretase modulators derived from the extract of Actaea racemosa.

A screen of a library of synthetic drugs and natural product extracts identified a botanical extract that modulates the processing of amyloid precursor protein (APP) in cultured cells to produce a lowered ratio of amyloid-beta peptide (1-42) (Aß42) relative to Aß40. This profile is of interest as a potential treatment for Alzheimer's disease. The extract, from the black cohosh plant (Actaea racemosa), was subjected to bioassay guided fractionation to isolate active components. Using a combination of normal-phase and reverse-phase chromatography, a novel triterpene monoglycoside, 1, was isolated. This compound was found to have an IC(50) of 100 nM for selectively reducing the production of amyloidogenic Aß42 while having a much smaller effect on the production of Aß40 (IC(50) 6.3 µM) in cultured cells overexpressing APP. Using IP-MS methods, this compound was found to modulate the pool of total Aß produced by reducing the proportion of Aß42 while increasing the relative amounts of shorter and less amyloidogenic Aß37 and Aß39. Concentrations of 1 sufficient to lower levels of Aß42 substantially (up to 10 µM) did not significantly affect the processing of Notch or other aspects of APP processing. When 1 (10 µg) was administered to CD-1 normal mice intracerebroventricularly, the level of Aß42 in brain was reduced. Assays for off-target pharmacology and the absence of overt signs of toxicity in mice dosed with compound 1 suggest a comparatively selective pharmacology for this triterpenoid. Compound 1 represents a new lead for the development of potential treatments for Alzheimer's disease via modulation of gamma-secretase.

Optimization of a natural product-based class of γ-secretase modulators.

A series of triterpene-based γ-secretase modulators is optimized. An acetate present at the C24 position of the natural product was replaced with either carbamates or ethers to provide compounds with better metabolic stability. With one of those pharmacophores in place at C24, morpholines or carbamates were installed at the C3 position to refine the physicochemical properties of the analogues. This strategy gave compounds with low clearance and good distribution into the central nervous system (CNS) of CD-1 mice. Two of these compounds, 100 and 120, were tested for a pharmacodynamic effect in the strain and lowered brain Aß42 levels.

Modulation of gamma-secretase for the treatment of Alzheimer's disease.

The Amyloid Hypothesis states that the cascade of events associated with Alzheimer's disease (AD)-formation of amyloid plaques, neurofibrillary tangles, synaptic loss, neurodegeneration, and cognitive decline-are triggered by Aß peptide dysregulation (Kakuda et al., 2006, Sato et al., 2003, Qi-Takahara et al., 2005). Since γ-secretase is critical for Aß production, many in the biopharmaceutical community focused on γ-secretase as a target for therapeutic approaches for Alzheimer's disease. However, pharmacological approaches to control γ-secretase activity are challenging because the enzyme has multiple, physiologically critical protein substrates. To lower amyloidogenic Aß peptides without affecting other γ-secretase substrates, the epsilon (ε) cleavage that is essential for the activity of many substrates must be preserved. Small molecule modulators of γ-secretase activity have been discovered that spare the ε cleavage of APP and other substrates while decreasing the production of Aß(42). Multiple chemical classes of γ-secretase modulators have been identified which differ in the pattern of Aß peptides produced. Ideally, modulators will allow the ε cleavage of all substrates while shifting APP cleavage from Aß(42) and other highly amyloidogenic Aß peptides to shorter and less neurotoxic forms of the peptides without altering the total Aß pool. Here, we compare chemically distinct modulators for effects on APP processing and in vivo activity.

Initial Optimization of a New Series of γ-Secretase Modulators Derived from a Triterpene Glycoside.

The discovery of a new series of γ-secretase modulators is disclosed. Starting from a triterpene glycoside γ-secretase modulator that gave a very low brain-to-plasma ratio, initial SAR and optimization involved replacement of a pendant sugar with a series of morpholines. This modification led to two compounds with significantly improved central nervous system (CNS) exposure.

Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist.

The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a "steric doorstop" holding the protein in an open and active conformation.

An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis.

Previous studies by Papermaster and coworkers introduced the use of rhodopsin-green fluorescent protein (rho-GFP) fusion proteins in the construction of transgenic Xenopus laevis with retinal rod photoreceptor cell-specific transgene expression [Moritz et al., J. Biol. Chem. 276 (2001) 28242-28251]. These pioneering studies have helped to develop the Xenopus system not only for use in the investigation of rhodopsin biosynthesis and targeting, but for studies of the phototransduction cascade as well. However, the rho-GFP fusion protein used in the earlier work had only 50% of the specific activity of wild-type rhodopsin for activation of transducin and only 10% of the activity of wild-type in rhodopsin kinase assays. While not a problem for the biosynthesis studies, this does present a problem for investigation of the phototransduction cascade. We report here an improved rhodopsin/EGFP fusion protein in which placement of the EGFP domain at the C-terminus of rhodopsin results in wild-type activity for activation of transducin, wild-type ability to serve as a substrate for rhodopsin kinase, and wild-type localization of the protein to the rod photoreceptor cell outer segment in transgenic X. laevis.