Targeted Polypharmacology: Discovery of a Highly Potent Non-Hydroxamate Dual Matrix Metalloproteinase (MMP)-10/-13 Inhibitor

Matrix metalloproteinases (MMPs) play a key role in many diseases like cancer, atherosclerosis or arthritis. Interest in MMP inhibition has been revitalized very recently as the knowledge on the underlying network of biological pathways is steadily growing. On the basis of this new insight into the relevance of MMP-10 and MMP-13 within the MMP network and the ban of hydroxamate inhibitors from clinical development, the discovery of non-hydroxamate multitarget drugs against specific MMPs is of foremost interest. Here, we disclose the discovery of a very potent and selective non-hydroxamate MMP-10/-13 inhibitor. The high potency (IC₅₀ of 31 nM [MMP-10] and 5 nM [MMP-13]) and selectivity over MMP-1, -2, -3, -7, -8, -9, -12, and -14 enable this compound to decipher disease causing MMP networks and to generate new treatment options through targeted polypharmacology

Additional file 3: of Conundrums in neurology: diagnosing serotonin syndrome – a meta-analysis of cases

List of 299 cases derived from 257 articles, reference list for data set. (DOCX 98 kb

Additional file 1: of Conundrums in neurology: diagnosing serotonin syndrome – a meta-analysis of cases

Method for meta-analysis of our cases (MAC) adapted to the PRISMA checklist, table. (DOCX 118 kb

Schematic representation of the proposed sequential and hierarchical control of multiple pancreatic transcription factors (pTFs) induced liver to pancreas transdifferentiation.

<p>Liver to pancreas transdifferentiation is a fast and sequential process which is temporally controlled in a hierarchical manner. Three transcription factors promote the transdifferentiation efficiency such that many more cells produce pancreatic hormones (B compared to A). However, to increase both transdifferentiation efficiency and the cells maturation along the β cell lineage (maturation means pro-insulin processing and its glucose regulated secretion), the 3 pTFs which control distinct stages of pancreas organogenesis should be sequentially supplied in a direct hierarchical mode (C).</p

Combined expression of pTFs promotes transdifferentiation efficiency, however only their sequential expression increases β-cell maturation.

<p>Cultured adult human liver cells were infected with <i>Ad-CMV-Pdx1</i> (1000 MOI), <i>Ad-CMV-Pax-4</i> (100 MOI) and <i>Ad-CMV-Mafa</i> (10 MOI) together or in a sequential manner as summarized in (A) (treatments B–E) or with control virus (<i>Ad-CMV-β-gal</i>, 1000 MOI, treatment A), and analyzed for their pancreatic differentiation six days later. (B) Immunofluorescence staining of treated human liver cells for insulin (red). Nuclei were stained with DAPI (blue), original magnification X20. The percent of insulin positive cells were calculated by counting at least 1000 positive cells from at least two independent experiments preformed in cells isolated from different donors. Insulin (and or pro-insulin) (C) and c-peptide (D) secretion were measured by static incubation of the cells for 15 min at 2 and 17.5 mM glucose in KRB. *<i>p<</i>0.05, **<i>p<</i>0.01, compared to control virus treated cells; n≥12 in 5 independent experiments preformed in cells isolated from different donors. (E) Quantitative Real-Time PCR analysis for gene expression of endogenous pTFs. CT values are normalized to β-actin gene expression within the same cDNA sample. Results are presented as relative levels of the mean±SE of the relative expression versus control virus treated cells. *<i>p<0.05</i>, n≥8 in 4 independent experiments preformed in cells isolated from different donors. The arrow points to the specific decrease in Isl1 expression level under the C-protocol, sequential and direct hierarchical administration of pTFs.</p

High Isl1 levels disrupt β cell maturation of transdifferentiated liver cells.

<p>Adult human liver cells were treated by the direct “hierarchical” sequential infection order (C) and supplemented by <i>Ad-CMV-Isl1</i> (1 or 100MOI) at the 3rd day (C+Isl1). <b>(A)</b> Quantitative Real-Time PCR analyses for insulin gene expression levels. CT values were normalized to β-actin gene expression within the same cDNA sample. Results are presented as relative levels of the mean ± SE compared to control virus treated cells. *P<0.05, n>6 in 3 independent experiments preformed in cells isolated from different donors. <b>(B)</b> Insulin (and or pro-insulin) secretion was measured by static incubation of the cells for 15 min at 2 and 17.5 mM glucose in KRB. **P<0.01, compared to the direct “hierarchical” sequential infection order (C). n>6 in 3 independent experiments preformed in cells isolated from different donors. <b>(C)</b> Quantitative Real-Time PCR analyses for the gene expression levels of SLC2A2. CT values are normalized to β-actin gene expression within the same cDNA sample. Results are presented as relative levels of the mean ±SE compared to control virus treated cells. *P<0.05, n>6 in 3 independent experiments preformed in cells isolated from different donors.</p

Mice with a hepatocyte-specific IFNAR ablation show infection of hepatocytes and severe hepatocellular necrosis.

<p>C57BL/6, IFNAR^-/-, Alb-Cre^+/-IFNAR^fl/fl, and LysM-Cre^+/-IFNAR^fl/fl mice were infected i.p. with 2 × 10⁴ PFU CVB3 and sacrificed 0, 2, or 3 dpi (n = 3–5). (A) Liver sections were H&E stained (line 1 and 2; bars = 10 μm) or subjected to CVB3-specific immunohistochemistry (line 3 and 4; bars = 50 μm). Arrowheads highlight necrotic hepatocytes (coagulative necrosis). Inserts show a lack of immunoreactivity in biliary ducts. (B) Quantification of area of infected liver tissue in CVB3-immunohistochemistry sections was performed by AnalySIS 3.2 software (n = 3–5). Bars depict median. Mann-Whitney test was used for statistical analysis, <i>*P < 0</i>.<i>05; **P < 0</i>.<i>01</i>.</p

During CVB3 infection abundant IFN-I and IFN-III responses are induced in liver and pancreas.

<p>IFN-β^wt/Δβ-luc mice (A-C) or C57BL/6 mice (D-F) were infected i.p. with 2 × 10⁴ PFU CVB3 and (A) luciferase activity was analyzed at the indicated time points by <i>in vivo</i> imaging. One representative mouse is shown. (B) Quantification of bioluminescence imaging in selected regions of interest (ROI) for upper abdominal (red) and cervical region (blue). Results are pooled from three independent experiments (n = 3–6). Values are mean ± SD. One-Way ANOVA test was used for statistical analysis. (C) IFN-β^wt/Δβ-luc mice were perfused with PBS at 0, 2, 4, and 7 dpi (n = 3–9). Spleen, cervical lymph nodes (cLN), liver, pancreas, salivary glands, and heart were prepared, homogenized, and the reporter activity was determined. Values are mean ± SD. Mann-Whitney test was used for statistical analysis. (D-F) Mice were sacrificed at 0, 1, 2, 3, 4, or 7 dpi (n = 3–6). Homogenates from liver and pancreas were assessed for (D) IFN-β, (E) IFN-α, and (F) IFN-λ protein levels by ELISA methods. Pooled data from two independent experiments are shown. Bars depict median. Mann-Whitney test was used for statistical analysis, <i>*P < 0</i>.<i>05; **P < 0</i>.<i>01; ***P < 0</i>.<i>001</i>.</p

<i>In vitro</i> CVB3 infected murine and human primary hepatocytes mount IFN-β responses that are essential for robust ISG induction.

<p>(A and B) <i>In vitro</i> cultured primary hepatocytes from C57BL/6 mice were CVB3 infected in triplicates at MOI 0.01, 0.1, or 1 and after 24 hr incubation supernatants and cells were harvested. (A) <i>Ifn-β</i>, <i>Ifn-α</i>, <i>Ifn-λ2/3</i> as well as <i>Isg15</i> mRNA expression levels were analyzed by qPCR and (B) IFN-β, IFN-α, and IFN-λ3 levels were determined from supernatants by ELISA methods. Pooled data from 4 similar experiments are shown (n ≥ 1). Bars depict mean + SEM. Dotted lines indicate the detection limits of 15.6 pg/mL IFN-β, 12.5 pg/mL IFN-α, and 30.625 pg/mL IFN-λ3. (C and D) Human primary hepatocytes were infected with CVB3 at MOI 0.1 or 0.3 and after 24 hr incubation supernatants and cells were harvested. (C) mRNA levels of <i>Ifn-β</i>, <i>Ifn-α</i>, <i>Ifn-λ1</i> as well as <i>Isg15</i> were analyzed by qPCR. (D) IFN-β, IFN-α, and IFN-λ1/2/3 levels were determined by ELISA methods. Pooled data from five similar experiments are shown. Bars depict mean + SEM. Dotted lines indicate the detection limits of 1.2 pg/mL IFN-β, 3.2 pg/mL IFN-α, and 62.5 pg/mL IFN-λ1/2/3. Primary human hepatocytes from donor 1, 2, 3, 4, and 5 were CVB3 infected at MOI 0.1 for 2 hr, washed with PBS, treated either with B18R, αFNLR, or both and incubated for 12 hr for donor 1–3 and 24 hr for donor 1–5. Cells were harvested and (E) Mx mRNA levels were analyzed by qPCR. (F) Total RNA of donor 1 and 2 after 24 hpi was additionally analyzed by RNAseq and the expression of 268 ISGs was studied. The induction of the 5 most abundantly upregulated ISGs are shown as [fold change] relative to uninfected controls in a heat map (left) and as a graph showing total expression levels in [Reads Per Kilobase of transcript per Million mapped reads, RPKM] (right).</p

Mice with a hepatocyte-specific IFNAR deletion succumb to CVB3 infection and show ubiquitous virus dissemination.

<p>C57BL/6 (black), IFNAR^-/- (white), Alb-Cre^+/-IFNAR^fl/fl (red), and LysM-Cre^+/-IFNAR^fl/fl (blue) mice were infected i.p with 2 × 10⁴ PFU CVB3 and (A) monitored for survival. Mice that showed severe clinical symptoms or more than 20% body weight lost were sacrificed (n = 9–16). Differences between the groups were determined using log-rank statistics. (B) Alanine aminotransferase (ALT) or lipase levels were determined from serum at the indicated dpi. Of note, for Alb-Cre^+/-IFNAR^fl/fl mice values at 4 and 5 dpi from only one surviving mouse per day are depicted. Values are mean ± SEM (n = 3–13). For statistical analysis, IFNAR^-/-, Alb-Cre^+/-IFNAR^fl/fl, or LysM-Cre^+/-IFNAR^fl/fl mice were compared with C57BL/6 mice by One-Way ANOVA test. (C) Graphs depict virus titers in liver, pancreas, spleen, heart, salivary gland (SG), and brain based on plaque formation 3 dpi (n = 2–5). Bars depict mean. Mann-Whitney test was used for statistical analysis, <i>*P < 0</i>.<i>05; **P < 0</i>.<i>01</i>. <i>***P < 0</i>.<i>001</i>.</p