WO2018074929A2

WO2018074929A2 - Drugs mimicking calorie restriction and tools and methods for identifying the same

Info

Publication number: WO2018074929A2
Application number: PCT/NL2017/050689
Authority: WO
Inventors: Cornelis Ferdinand CALKHOVEN
Original assignee: Rijksuniversiteit Groningen; Academisch Ziekenhuis Groningen
Priority date: 2016-10-20
Filing date: 2017-10-20
Publication date: 2018-04-26
Also published as: WO2018074929A3

Abstract

The invention relates to the fields of medicine and drug development, more particularly to drugs that are capable of mimicking calorie restriction (CR), and to screening tools and screening methods for identifying the same. Provided is a dual reporter plasmid system comprising a first nucleic acid sequence encoding a Renilla luciferase transcript and a second nucleic acid sequence encoding a firefly luciferase transcript, wherein said nucleic acid sequences are both under control of the C/EBPβ-5'UTR-uORF. Also provided is adefovir or a prodrug thereof for use in a method for improving the metabolic health in a subject.

Description

Title: Drugs mimicking calorie restriction and tools and methods for identifying the same.

The invention generally relates to the fields of medicine and drug development. More particularly, it relates to drugs that are capable of mimicking calorie restriction, and to screening tools and screening methods for identifying the same. Whereas mammals have widely differing lifespans, a common feature of lifespans is that they seems to be adjustable and can be increased by genetic or pharmacological interventions. Dietary calorie restriction (CR) (also called dietary restriction) is the best-documented intervention that delays most age-related physiological changes, and extends life span in all species tested, provided malnutrition is avoided (Weindruch, et al. The Retardation of Aging and Disease by Dietary Restriction (Charles C.

Thomas, Springfield, 111., 1988)). CR has been shown to potentially increase hfespan by 25-35% in all animals studied to date (mice, rats, several species of monkeys, dogs₇ as well as non-metazoan species such as spiders,

Nematodes, and Drosophila). These studies also have shown that CR is a highly, if not the most, effective means now known for reducing cancer incidence and increasing the mean age of onset of age-related diseases and tumors in homeothermic vertebrates (Weindruch et al. (1982) Science 215: 1415). Thus, it seems clear that hfespans can be extended through a relatively simple dietary regimen.

Several genes in mice have been identified as "longevity" genes because mice with mutations in these genes have greater mean lifespans relative to the expected lifespan of control mice. These genes include the Ames dwarf mutation, and the Snell dwarf mutation. However, these mutations result in small, frail mice which have difficulty feeding. Therefore, it is beheved that the longevity conferred by these mutations is essentially due to calorie restriction. Recent attempts to use gene array analysis, or other genetic screens for genes associated with longevity phenotypes in worms, flies, and rodents have come up with a number of candidate genes. In general, however, they are frequently "stress-response" genes.

Sirtuins are highly conserved protein deacetylases and/or ADP- ribosyltransferases that have been shown to extend lifespan in lower model organisms, such as yeast, C. elegans, and drosophila. In mammals, sirtuins have been shown to act as metabolic sensors, responding to environmental signals to coordinate the activity of genes that regulate multiple energy homeostasis pathways. For example, studies have shown that sirtuin activation mimics the effects of caloric restriction, an intervention

demonstrated to significantly extend lifespan, and activates genes that improve glucose homeostasis and the conversion of fat to energy by fatty acid oxidation (Guarente, 2012, Nature Reviews Molecular Cell Biology 13, 207).

Some compounds, such as gingko, ginseng and vitamin C, have been proposed to improve survival, but controlled and statistically significant survival studies reporting the benefit for these compounds are unknown. Vitamin C and a number of drugs reduce the incidence of certain disease conditions, e.g. cardiovascular disease, and so, presumably, would enhance overall longevity. It is also known that an important part of the beneficial effects of CR on health- and life-span is mediated through regulation of protein synthesis that is under control of the nutrient sensitive mechanistic target of rapamycin complex 1 (mTORC l). mTORC l signaling coordinates the regulation of global protein synthesis and autophagy to adapt protein homeostasis to changing nutrient availability and growth factor signaling (Shimobayashi and Hall, 2014). In addition, the expression of a subset of proteins is specifically regulated by mTORC 1 through distinctive

translation of their mRNAs involving cis-regulatory elements that make them responsive to regulators of translation. Previously, we presented that specific translation into the C/ΕΒΡβ protein isoform LIP (Liver-specific Inhibitory Protein) is under control of mTORC 1 trough regulation of the downstream eukaryotic translation initiation factor 4E (eIF-4E) binding proteins (4E-BPs), and that mTORC 1 -inhibition by rapamycin reduces LIP expression (Calkhoven et al., 2000; Zidek et al., 2015).

Translation of the C/EBP -mRNA involves two separate translation mechanisms, initiation and re-initiation: 1) Synthesis of the isoforms LAP and LAP* (Liver-specific Activating Protein) is the result of regular translation initiation where ribosomes scan the mRNA from the 5'-end to the first AUG-codon in a favorable Kozak sequence context to initiate translation (LAP* is often weakly expressed because it has no Kozak sequence); 2) translation into LIP requires the initial translation of a cis- regulatory upstream open reading frame (uORF) in the mRNA leader- sequence, followed by the continuation of mRNA scanning and translation re-initiation from the downstream LIP-AUG coclon (Calkhoven et al., 2000). Activated mTORC 1 signahng stimulates the latter re-initiation into LIP but only marginally affects the initiation into LAP. The dependence of LIP expression on the presence of the uORF can be explained by the finding that translation of the uORF prevents translation initiation at the LAP initiation codon. Mechanistically, this is based on the fact that uORF -post-termination ribosomes have to be reloaded with new initiator tRNA (Met-tRNAi Met) in order to perform a second translation re-initiation at the same mRNA molecule. The LAP initiation codon is too close (4 nt) to the uORF for the post-termination ribosomes to be reloaded with new Met-tRNAi^{M i} in time. Therefore, they omit the LAP initiation codon but can be reloaded with Met- tRNAi^Met early enough to re-initiate translation at the downstream LIP initiation codon. The sophisticated structure of the C/EBP -mRNA renders its translation responsive to changes in availability or activity of

translation-regulatory factors.

Thus, as one of its activities, mTORCl stimulates the expression of the metabolic transcription factor CCAAT/Enhancer Binding Protein β

(C/ΕΒΡβ) isoform Liver-specific Inhibitory Protein (LIP). Regulation of LIP expression strictly depends on a translation re-initiation event that requires a conserved cis-regulatory upstream open reading frame (uORF) in the C EBP -mRNA. We have shown recently that experimental suppression of LIP in mice, reflecting reduced mTORCl -signaling at the C/ΕΒΡβ level, results in CR-type of metabolic improvements. (Zidek et al (2015) EMBO Rep. 16, 1022-1036). However, means or methods to reduce C/EBP6-LIP levels with the aim to mimic CR are at present not available.

Recognizing the therapeutic potential of compounds capable of modulating levels of LIP, the present inventors set out to search for novel possibilities to pharmacologically down-regulate LIP in order to induce CR-mimetic effects. To that end, they developed and performed a screening strategy for the identification of compounds that alter C/EBP -uORF dependent translation re-initiation into LIP. The screening strategy involved a novel reporter assay system (see herein below), which was validated with pharmacological and genetic approaches. The screening of a library of FDA approved drugs surprisingly revealed Adefovir Dipivoxil as a single drug that reduces translation re-initiation in different cell lines, reduces endogenous LIP expression and increases β-oxidation in a mouse hepatoma cell line. Herewith, the invention provides adefovir, or a prodrug thereof, for use in a method for improving the metabolic health in a subject. As used herein, the term improving metabolic health' refers to all aspects relating to CR, including increase β-oxidation, improved insulin sensitivity and glucose tolerance, reduced body weight, reduced fat starage, prevention of steatosis (fatty liver), reduced serum lipids. Also comprised is body weight-related cancer incidence.

In one embodiment, improving the metabolic health comprises shifting the metabolism towards enhanced beta-oxidation. In another embodiment, improving the metabolic health comprises reducing weight gain or enhancing or supporting weight loss in a subject.

Preferably , the subject is a human subject. Of particular interest are elderly subjects or subjects suffering from, or at risk of developing, a condition selected from the group consisting of metabolic syndrome, obesity (foiexample, visceral fat accumulation, subcutaneous fat accumulation), type 2 diabetes, pre-diabetes, hypertension, disorders of lipid metabolism (for example, fatty liver, hyperlipidemia, dyslipidemia), insulin resistance, endothelial dysfunction, pro-inflammatory state, and pro-coagulative state.

However, the invention is also suitably used to improve the metabolic health in a non-human mammalian subject, like a pet or a husbandry animal.

Also provided herein is adefovir or an adefovir prodrug for use as a calorie restriction mimetic compound.

In a preferred embodiment of the invention, the adefovir prodrug is adefovir dipivoxil. Adefovir dipivoxil was approved in 2002 for the treatment of chronic hepatitis B. HEPSERA™ is the tradename for adefovir dipivoxil, a diester prodrug of adefovir. Adefovir is an acyclic nucleotide analogue of adenosine monophosphate that inhibits the hepatitis B virus (HBV) DNA polymerase by competing with the natural substrate deoxyadenosine triphosphate and by causing DNA chain termination after its incorporation into viral DNA. The chemical name of adefovir dipivoxil is 9-[2- [bis[(pivaloyloxy)methoxy] phosphinyl]methoxy] -ethyl] adenine. Adefovir is phosphorylated to the active metabolite, adefovir diphosphate, by cellular kinases.

A further embodiment relates to a weight management composition comprising adefovir or an adefovir prodrug, e.g. adefovir dipivoxil, in combination with one or more further active ingredient(s) supporting or enhancing weight loss which is known in the art or which is yet to be discovered.

In one embodiment, the composition comprises, in addition to adefovir or an adefovir prodrug, a weight loss effective amount of a noradrenaline stimulating compound such as ephedrine, mahuang (a plant source of ephedrine alkaloids), citrus aurantium (bitter orange), synephrine, norephedrine, psuedophedrine, a methylxanthine, such as caffeine or guarana, and/or a COX-2 inhibitor such as resveratrol, polygonum

cuspidatum, Scutellaria baicalensis, white willow bark, turmeric, curcumin, rosemary, green tea, ocimum sanctum (holy basil), or ginger. See US

6,475,530.

In another embodiment, the composition comprises a biologically effective amount of an extract of a plant of the genus Eucalyptus as an active ingredient, which extract was found to be useful in a method of inhibiting or preventing obesity (increase in weight) lipid storage disease,

hyperlipidemia, arteriosclerosis and thrombosis, and a method of inhibiting or reducing an amount of triglyceride in blood and an amount of cholesterol in blood (see US2007/0054000A1). In a further embodiment of the invention, adefovir or an adefovir prodrug, e.g. adefovir dipivoxil, is combined with (a) at least a kind of polyphenols selected from the group consisting of hesperidin, a hesperidin derivative, and hesperetin; and (b) at least one xanthine derivative, the mass ratio of (a):(b) in the composition being 1:(0.001 to 5). See US2015/0265645A1.

In yet another aspect, the invention relates to screening tools and screening methods for identifying the further compounds that, like adefovir, are capable of mimicking calorie restriction.

Conditions that reduce LIP levels, like CR or other means of mTORC 1 inhibition, are currently being explored for its feasibihty to improve metabolic health and to treat cancer. Long term CR, however, is not manageable for most people and current treatment with mTORC 1 inhibitors are of limited success and come with serious side effects (Stallone et al., 2009; Wander et al., 2011; Willemsen et al., 2016; Zaytseva et al., 2012). Transcription factor function is considered to be largely undruggable and, in case of LIP, it would almost inevitably mean that LAP is targeted too, since LAP includes all sequences that make up LIP.

As described herein above, the present inventors recognized that the specific translation mechanism that is responsible for LIP translation is potentially draggable, as reduction of LIP through pharmacological inhibition of mTORC 1 exemplifies.

Therefore, they set out to develop a screening strategy for the identification of compounds that alter C/EBPp-uORF dependent translation re-initiation into LIP and thereby ultimately identify compounds that have CR-mimetic and or anti-cancer activities. Provided herein is a cellular system containing C/EBP -uORF controlled luciferase-reporters that measures simultaneously translation initiation and re-initiation and is suited for high-throughput screening strategies. More specifically, the invention provides a dual reporter plasmid system comprising a first nucleic acid sequence encoding a Renilla luciferase transcript and a second nucleic acid sequence encoding a firefly luciferase transcript, wherein said first and second nucleic acid sequence are both under control of the C/EBP -5'UTR- uORF (upstream Open Reading Frame). Whereas reporter systems based on C EBPa-uORF regulation have been used in the past (Wiesenthal et al, 2006a; Wiesenthal et al, 2006b), these systems use a different uORF.

Moreover, they are based on immunotags and require Western blot analyis or ELISA-type assays, which are not suitable for HTS applications.

The dual reporter plasmid system of the invention comprises 1) a translation initiation cassette with a nucleic acid sequence encoding a renilla luciferase transcript as surrogate for C/EBPp-LAP expression and 2) a translation re-initiation cassette with a nucleic acid sequence encoding a firefly luciferase transcript as surrogate for ϋ/ΕΒΡβ-ΚΙΡ expression, wherein said first and second nucleic acid sequence are both under control of the C/EBP -5'UTR-uORF. See Figure lA for a schematic drawing.

Herewith, the invention provides a cellular reporter system as a surrogate for measuring the ratio between translation initiation and uORF-dependent re-initiation as it is used as a mechanism for differential translation of the C/EBP -mRNA into the protein isoforms LAP and LIP.

The system we present here belongs to the type of phenotypic drug discovery (forward pharmacology) where compounds are identified that cause a desirable change in phenotype, here reduction of C/EBP -uORF- dependent translation re-initiation. The system, however, is not purely phenotypic and target-agnostic (without having prior knowledge of their biological activity or mode of action against a specific molecular target or targets) and is better described as a translation re-initiation and initiation mechanism-informed phenotypic screen (Moffat et al, 2014; Swinney & Anthony, 2011). Phenotypic screens potentially can identify compounds that modify a (disease) phenotype by acting on a yet undiscovered target or targets. The successive identification of the target(s) using for example techniques as chemical proteomics (Rix & Superti-Furga, 2009) could give novel insights into translation control mechanisms and involved upstream signaling pathways and might thereby also broaden our knowledge of potential players in metabolic diseases, aging and cancer.

Also provided is a cell comprising a dual reporter plasmid system according to the invention. Preferably, the plasmid is stably integrated into the genome of the cell. Suitable cells include mammahan cells or cell lines, like HEK293T, NIH-3T3, primary hepatocytes or iPS-derived cell types.

In one embodiment, a cell comprising the dual reporter system of the invention finds its use in drug-development. For example, provided herein is a method or an assay for identifying a compound capable of mimicking calorie restriction (CR) and/or having anti-cancer activity, comprising the steps of providing a cell culture comprising cells according to the invention, contacting said cell culture with a candidate compound (to allow for luciferase expression), determining expression oiRenilla luciferase and firefly luciferase, calculating the translation re-initiation index (TRI) based on the formula

TRI = Firefly ^{Re hliiiati011} x ReniHa^Reference / Renilla^{lni1i tion} x Firefly^Reference and correlating said TRI with the capacity of said compound to mimic calorie restriction, wherein a reduction in TRI is a positive indicator of the compound being capable of mimicking calorie restriction (CR) and or having anti-cancer activity. In one embodiment, determining expression of Renilla luciferase and firefly luciferase comprises the quantitation of a stable luminescent signal from two reporter genes in a single sample. For example, commercial kits are available which provide a convenient "add-and-read" system that generates both firefly and Renilla luciferase luminescence signals from cells that have not been preconditioned or prelysed An additional reporter plasmid may be used for measurement of reference levels of firefly (FireflyRef) and renilla (RenillaRef) luciferase through unrestrained translation (Fig. IB, pcDNA FireflyRef/RenillaRef). This reporter can serve for the counter assay to control for effects not related to reinitiation/initiation efficiency, like the rate of transcription, mRNA stabilization, general effect on translation and/or direct interference with the luciferase activity. Accordingly, in one embodiment the assay or method further comprises cell comprising a second reporter reference plasmid system, comprising a first nucleic acid sequence encoding a renilla luciferase transcript and a second nucleic acid sequence encoding a firefly luciferase transcript without C/EBP -5'UTR-uORF sequences (see Figure 1 B).

Also provided herein is a kit-of-parts comprising a dual reporter plasmid system according to the invention, a luciferase substrate, and optionally a reference reporter plasmid as mentioned herein above.

A method and/or kit of the invention is advantageously used in a high throughput drug screening assay, in particular for the identification of compounds capable of improving the metabolic health in a subject.

LEGEND TO THE FIGURES

Figure 1. Translation Re-initiation Index (TRI) determination.

A) Representation of pcDNA3-Firefly^{Re illi}/Renilla^lni plasmid containing the initiation and re-initiation cassette as indicated. Renilla luciferase (Renilla¹¹¹¹) can only be translated by ribosomes that have scanned over the uORF and directly initiate at the AUG initiation codon of the renilla reading frame. Translation of the firefly luciferase (Firefly^Re' ^li) is only possible by ribosomes that have first translated the uORF, resume scanning and re-initiate at the AUG initiation codon of the firefly reading frame. The latter transcript also contains a "dummy" reading frame that captures those ribosomes that have bypassed the uORF to directly initiate at the "dummy" AUG initiation codon. Here, no signal is produced.

B) Representation of pcDN A3 -Firefly ^R('^r/Renilla^{R r} plasmid for measurements of reference levels of firefly luciferase and renilla luciferase . The formula used for calculation of the translation re-initiation index (TRI) is shown framed.

Figure 2. Reporter system validation.

A) Mouse embryonic fibroblasts (MEFs) that are deficient in the mTORC 1 -inhibitor protein TSC 1 (TSC 1-KO) or wt MEFs transiently transfected with pcDNAS-Firefly^-^^/Renilla¹¹¹' and pcDNA- Firefly^Rei7Renilla^Rei reporter plasmids. The bar graphs show the calculated TRI mean values. Immunoblots show the levels of C/ΕΒΡβ, TSC l, phospho- S6K, S6K and the β-actin loading control.

B) 4E-BP 1/2 double knockout (DKO) MEFs and wt MEFs transiently transfected with reporter constructs as described in (a). The bar graphs show the calculated TRI mean values. Immunoblots show the levels of C/ΕΒΡβ, 4E-BP 1, 4E-BP2 and the β-actin loading control. C) HEK293T cells stably expressing pcDNA3-Firefly^Rf -ⁱⁿVRenilla^Ini or pcDNA-Firefly^Ref ReniIla^Ref reporters treated with 200 nM rapamycin or 10 nM PP242 for 8 h. The bar graphs show the calculated TRI mean values. The immunoblots show mTORC 1 inhibition by reduced phosphorylation of 4E-BP1 and S6K in addition to β-actin as loading control.

D) HEK293T cells described in (c) transfected with elF expression vectors. The bar graphs show the calculated TRI mean values. Immunoblots show levels of eIF4, eIF2a, PKR and the a-actin loading control.

E) HEK293T cells described in (c) treated with the PKR inhibitor (C16) for 8 h. The bar graphs show the calculated TRI mean values.

Immunoblots shows the reduced phosphorylation of eIF2a and the β-actin loading control.

F) DENR-KD (+Dox) and DENR-Control (-Dox) HEK293T cells transiently transfected with reporter constructs as described in (a). The bar graphs show the calculated TRI mean values. Immunoblots show levels of DENR and the β-actin loading control.

G) C33A cells with stable SBDS knockdown (shSBDS) or control cells (shCTRL) transiently transfected with reporter constructs as described in (a). The bar graphs show the calculated TRI mean values. Immunoblots show levels of SBDS and the β-actin loading control. Statistical differences were analyzed by Student's t-tests. Error bars represent ±SD (n = 5), *P < 0.05, **P < 0. 01, ***P < 0. 001.

Figure 3. High throughput screening of a drug library.

A, B and C) Scatter plots for three different plates with measured translation re-initiation/initiation ratio (Firefly^{Re ini}/Renilla^Ini) of 780 FDA- approved drugs. Each plate contained 6 wells treated with DMSO as a solvent and rapamycin as a positive control (bar graph at the right, error bars represent SD). The threshold of three times the standard deviation from the DMSO mean value (3xSD) is indicated. Drugs that decreased the translation re-initiation/initiation ratio more than 3xSD are indicated in red. Drugs indicated by numbers (#1, 2, 3 and 4) were selected for further evaluation. The arrows point to Sirolimus in plate 1 and Everolimus in plate 2.

D) Names and structural formulas of the drug #1-4 (retrieved from the ChemSpider database, www.clieinspider.coin). Statistical differences were analyzed by Student's t-tests. Error bars represent ±SD (n = 6), **P < 0.01, ***p < 0. 001. Figure 4. Validation of the drugs that decrease translation reinitiation/initiation ratio.

A) TBI mean values of HEK293T cells transiently transfected with pcDNA3-FireflyRe-ini/RenillaIni and pcDNA3-FireflyRef/RenillaRef reporter plasmids and treated with drug #1, 2, 3 or 4.

B and C)TRI mean values of Hepal-6 or MCF7 cells transiently transfected with the constructs described in (A) and treated with drugs #2, 3 or 4.

D) Treatment with drug #4 (adefovir dipivoxil; 10 μΜ for 8 h) decreases C/EBPp-LIP/LAP ratio in HEK293T cells as analyzed by immunoblotting. β-actin was used as a loading control. Bar chart at the right shows quantification of the relative changes in LIP/LAP -isoform ratio by adefovir dipivoxil and rapamycin compared to DMSO solvent.

E and F)Same experiment as described in (D) with Hepal-6 or MCF7 cells, respectively. C/ΕΒΡβ -LIP/LAP isoform ratios were quantified by chemiluminescence and digital imaging. Statistical differences were analyzed by Student's t-tests. Error bars represent ±SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0. 001. Figure 5. Adefovir dipivoxil augments fatty acid oxidation.

Increased oxygen consumption rate (OCR) upon usage of palmitate as exogenous energy source (fatty acid oxidation) in Hep a 1-6 cells treated with adefovir dipivoxil (#4). Panel A: OCR without palmitate-BSA. Panel B: OCR with palmitate-BSA, the latter being abolished by ectopic expression of C/EBPp-LIP (panel B, right bar graph). Statistical differences were analyzed by Student's t-tests. Error bars represent ±SD (n = 5), **P < 0.01.

EXPERIMENTAL SECTION Materials and Methods

DNA constructs

For generation of pcDNA-Firefly^R('"^kli/ReniHa^Ini, we first cloned the rat C EBPp-5'UTR until the LAP initiation codon in frame with the renilla sequence (from pGL4) in pcDNA3, and separately cloned C/EBPp-sequences spanning the 5'UTR and sequences until the LIP initiation codon with a +1 frame shift (7 nt upstream of the AUG) with the firefly sequences (from pGL3) in pcDNA3. A fragment of the pcDNA3-LAP-Renilla between the Bglll restriction site and spanning the renilla sequences was amplified by PCR using the following primers, forward: 5'-CGG AAA TGT TGA ATA CTC ATA CTC-'3 and reverse: 5'-GCT CAG ATC TCC TCA GAA GCC ATA GAG C-'3. The amplified fragment was sequenced and cloned in the pcDNA3-LIP- Firefly using the Bglll site to obtain the final pcDNA-Firefly^{Re ini}/Renilla^Ini. For the reference construct pcDNA-Firefly^Kef/ReniHa^Ref, a similar cloning strategy was employed using solely renilla and firefly coding regions (from pGL4 and pGL3 respectively). eIF4E, eIF2a-S51D, eIF2a-S5lA and PKRA6 pcDNA3-based expression vectors are described in (Calkhoven et al, 2000). The human DENR shRNA expression vector was generated by annealing the oligonucleotides shDENR /ora;ard:'5-CCG GCA AGA GTA TGT GGC CTT GCA ACT CGA GTT GCA AGG CCA CAT ACT CTT GTT TTT T-3' and shDENR reverse '5-AAT TAA AAA ACA AGA GTA TGT GGC CTT GCA ACT CGA GTT GCA AGG CCA CAT ACT CTT G-3' and ligating them into the Tet-pLKO-puro vector (Addgene plasmid #21915, described in

(Wiederschain et al, 2009)).

Cell culture

HEK293T and Hepal-6 cells were maintained in DMEM and MCF7 cells in RPMI (Gibco) supplemented with 10% fetal calf serum (FCS) and 1%

Penicillin/Streptomycin at 37°C with 5% C02. TSCl-KO and 4E-BP1/2-DKO MEFs were previously described(Le Bacquer et al, 2007; Zhang et al, 2003). SBDS-KD C33A cells were used before (In et al, 2016). Hepal-6 cells ectopically expressing C/EBP -LIP were described at(Zidek et al, 2015). Cell number and viability was determined using the TC20™ automated cell counter (Bio-Rad) following the manufacturers instruction. For generation of cells stably expressing the reporter constructs, pcDNA-Firefly^R('" lⁱ/Renilla^lni or pcDNA-Firefly^Rei/Renilla^Rei were transfected into HEK293T cells using FUGENE (Roche). Cell clones were selected by addition of G418 to the medium (1 mg/ml). For knockdown of DENR in HEK293T cells, the cells were transduced with the inducible lentiviral Tet-pLKO-puro-shDENR followed by selection of the transduced cells with puromycin (1.5 μg/ml). Then these cells were treated with doxycycline (100 ng/ml)for 36 h.

Rapamycin (R-5000, LC Laboratories), PP242 (#P0073, Sigma) and eIF-2a- kinase PKR inhibitor (C16) (#19785, Sigma) were used as chemical inhibitors.

Luciferase assay

For the Luciferase assay, 25000 cells per well were seeded in 96-well plates. After 24 h, cells were cotransfected with the plasmids as indicated using FUGENE HD (Promega), or stably transfectecl cells were used. The next day, luciferase activity was measured in cells treated with 8h of drug treatment or DMSO solvent control by Dual-Glo Luciferase Assay System (#2920, Promega) following the manufacturer's protocol using a GloMax- Multi Detection System (Promega).

Viability Assay

For the viability assay, 40000 HEK293T cells were seeded in 96-well plates. After 24h, cells were treated with the different drugs for 8h and cell viability was measured by Cell-Titer Flour cell viability assay system (#G6081, Promega) following the manufacturer's protocol using a GloMax-Multi Detection System (Promega).

Immimoblotting

For protein extraction, the cells were washed twice with ice-cold lx PBS and lysed in 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, supplemented with protease and phosphatase inhibitors followed by sonication. Equal amounts of total protein were separated by SDS-PAGE (#456-1094, BIO-RAD), transferred to a PVDF membrane using Trans-Blot Turbo System (#170-4273, BIO-RAD) following the manufacturer's protocol. The following antibodies were used: C/ΕΒΡβ (C19) and SBDS (S-15) from Santa Cruz Biotechnology; phospho-p70S6K (Thr389) (108D2), p70S6K (#9202), Hamartin/TSCl (D43E2) (#6935), phospho-4E-BPl (Thr37/46) (#9459), 4E-BP1 (#9452), 4E-BP2 (#2845), eIF4E (#9742), Phospho-eIF2a (Ser51) (#9721), eIF2a (#9722) and PKR (#3072) from Cell Signaling; DENR (#10656- 1-AP) from Proteintech™ and β-actin (clone C4) (#691001) from MP Biomedicals. HRP-conjugated secondary antibodies were purchased from Amersham Life Technologies. The bands were visuahzed by

chemiluminescence (ECL, Amersham Life Technologies) using ImageQuant LAS 4000 mini imaging machine (GE Healthcare Bioscience AB) and the supplied software was used for the quantification of the bands.

Library screening

HEK293T cells stably expressing Firefly^R('"™ Renilla^Ini vector were seeded at a density of 1.5xl0⁴ in 30 μΐ media per well of a 384-well plate (Greiner CELLSTAR® 384 well plates, white) and grown overnight. Next day, the cells were treated for eight hours with 780 FDA-approved drugs library (#BML-2843-0100, Enzo Lifescience) (final concentration 10 μΜ), 0.5% v/v DMSO and rapamycin (final concentration 200 nM) using an Echo® 555 Liquid Handler (Labcyte) transferring up to 150 nl to each well. After lysis of the cells, luciferase activity was measured.

Measurements of cellular oxygen consumption rate and fatty acid oxidation

Oxygen consumption rate and fatty acid oxidation were determined using a Seahorse XF96 Extracellular Flux analyzer (Seahorse Bioscience). For fatty acid oxidation assay, 2 l0 Hepa 1-6 cells per well were seeded into a 96- well XF cell culture microplate 24 h prior to the assay and cultured in the presence of 0.5 mM carnitine. Sixteen hours before the assay, cells were treated with the drugs. One hour before the assay, the cells were washed twice with FAO assay buffer (Seahorse Bioscience), and 15 min before the assay, the fatty acid oxidation substrate palmitate-BSA or a BSA control (Seahorse Bioscience) was added and the oxygen consumption rate (OCR) with or without palmitate-BSA was measured.

EXAMPLE 1: Reporter system to emulate (monitor) C/EBPJi-mRNA translation

In order to identify small molecule compounds that modulate

C/EBP -uORF dependent and differential translation of the C/EBPp-mRNA into the protein isoforms LAP and LIP we designed a reporter system as a surrogate for this process. A single dual reporter plasmid was constructed that expresses a renilla luciferase transcript for the simulation of

translation initiation into LAP and a firefly luciferase transcript for the simulation of translation re-initiation into LIP, both under control of the C/EBPB-5'UTR (Fig 1A, pcDNA-Firefly^Ri -""/Renilla¹"¹). Comparable to translation into LAP from the genuine C/EBPp-niRNA, regular translation initiation after leaky scanning over the uORF results in expression of renilla luciferase from the initiation cassette (Renilla'¹¹') (Fig 1A, Initiation

Cassette). This cassette does not contain the downstream LIP-AUG and therefore uORF translation and eventual subsequent downstream reinitiation events are not detected. The efficiency of uORF-dependent reinitiation, simulating translation into LIP, is measured as expression levels of firefly luciferase from the re-initiation cassette (Firefly ^{R illi}) (Fig 1A, Re- initiation Cassette). Ribosomes omitting translation of the uORF in the reinitiation cassette initiate translation at the proximal ORF consisting of C ΕΒΡβ sequences that would mimic translation at the LAP initiation site (Fig 1A, grey box), however, its product is not detected with the luciferase assay. In this way, only re-initiation is measured and direct initiation is left unnoticed. Alterations in the ratio between re-initiation and initiation are depicted as Firefly^{Re im}/ReniUa^Im values for the compound library screens.

An additional reporter plasmid was constructed for measurement of reference levels of firefly (Firefly^Rei) and renilla (Renilla^{Rf l} luciferase through unrestrained translation (Fig IB, pcDNA-Firefly^Rfif/Renilla^Ref)- This reporter is used for the counter assay to control for effects not related to reinitiation/initiation efficiency like the rate of transcription, mRNA

stabilization, general effect on translation or direct interference with the luciferase activity. Using measurements obtained with the pcDNA3- Firefly^{Ril ini}/Renilla^Ini and the pcDN A3 -Firefly ^VReniHa^ plasmids a translation re-initiation index (TRI) can be calculated by using the formula TRI = FirefLy^R<' ⁱⁿⁱ Renilla^Ref / Reiiilla^lni Firefly™ (Fig IB, boxed formula). Hence, an TRI > 1 indicates enhanced re-initiation and an TRI < 1 indicates reduced re-initiation, respectively. TRI provides a more stringent

normalized measurement for the validation of compounds identified by Firefly^Re"ⁱⁿⁱ/Renilla^lni reporter system.

EXAMPLE 2: Validation of the reporter system

The differential translation of the C/EBPp-mRNA into LAP and LIP protein isoforms is under control of the mTORCl/4E-BP/eIF4E pathway (Calkhoven et al, 2000; Zidek et al, 2015). To examine the performance of the reporter in response to mTORCl hyperactivation we separately transfectecl the pcDNA-Firefly^Rf' ⁱⁿVReniUa^Im and pcDNA-Firefly^Rei7ReniUa^R<!f reporter plasmids in mouse embryonic fibroblasts (MEFs) that are deficient in the mTORCl -inhibitory protein TSCl (TSCl-KO) (Kwiatkowski et al, 2002) or in wt MEFs and calculated the TRI. Compared to the wt MEFs the TRI was increased in the TSCl-KO MEFs, revealing enhanced translation re-initiation, which also resulted in enhanced expression of LIP (Fig 2A). A significant part of the mTORCl -dependent LIP regulation is mediated through the inhibitory phosphorylation of 4E-binding proteins (4E-BPs) by mTORCl, resulting in release of the eukaryotic translation initiation factor 4E (eIF-4E) (Zidek et al, 2015). In accordance, we found higher TRI values and increased LIP expression in 4E-BPl/2-double knockout (4E-BP1/2-DKO) MEFs comparing to wt MEFs (Fig 2B). mTORCl signaling can be

pharmacologically inhibited by the allosteric inhibitor rapamycin or the catalytic inhibitor PP424. To examine the performance of the reporter under pharmacological conditions we generated HEK293T based cell hnes with stable integrated pcDNA-Fire%^{Re in}VRenilla^[ni or pcDNA-Firefly^Ril|7Renilla^R('^f reporters. Treatment of the reporter cell lines with 200 nM rapamycin or 10 iiM PP242 decreased LIP expression and lowered TRI values, revealing suppression of re-initiation under inhibited mTORCl conditions (Fig 2C). The mTORCl inhibition was monitored by reduced phosphorylation of 4E- BP1 and S6 kinase (S6K). Finally, overexpression of eIF-4E that increases LIP expression accordingly increased TRI values (Fig 2D). Therefore, the reporter system reliably reflects differential translation of the C/ΕΒΡβ- mRNA under changing mTORC 1 signaling.

The eIF2a-kinases suppress post-uORF translation re-initiation through inhibitory phosphorylation of eIF2a in response to various stresses (Aitken & Lorsch, 2012; Sonenberg & Hinnebusch, 2009), which results in reduced expression of LIP (Calkhoven et al, 2000). Augmenting eIF2 activity by treatment with the eIF2a-kinase inhibitor C16, which reduces in eIF2a- phosphorylation, resulted in elevated translation re-initiation measured as higher TRI values and in an increase in LIP expression (Fig 2E). Similarly, activation of eIF2a by overexpression of the dominant negative mutant of the eIF2a-kinase PKR (PKRAG) or the non-phosphorylatable eIF2 S5lA mutant but not the phosphorylation mimicking eIF2aS51D mutant resulted higher TRI values (Fig 2D). Thus, the alterations in eIF2a-kinase signaling can be measured using the TRI reporter system.

In addition to the canonical translation initiation pathways we examined the effects of two proteins that were shown to determine the efficiency of translation re-initiation by different mechanisms. The density- regulated protein (DENR) has been identified as a key regulator of eukaryotic re-initiation downstream of uORFs, and knockdown of DENR impairs the post-uORF re-initiation of translation (Schleich et al, 2014). To examine the effect of DENR expression we generated HEK293T cells with a doxycycline-inducible DENR-shRNA expression vector. Induction of DENR- KD (+Dox) resulted in lower TRI comparing to control cells (Fig 2F), confirming the crucial role of DENR in uORF-dependent translation reinitiation. Recently, we have shown that deficiency in the Shwachman- Bodian-Diamond SyndiOme (SBDS) protein impairs translation re-initiation into the Ο/ΕΒΡβ-LIP isoform (In et al, 2016). In accordance, we measured lower TRI values in cells with shSBDS mediated knockdown of SBDS (Fig 2G).

Taken together, the TRI reporter system reliably detects changes in the ratio of initiation versus re-initiation by known translationally active drugs or alterations in key translation initiation factors and their regulatory kinases or non-canonical regulators of re-initiation.

EXAMPLE 3: Assay development and execution of a high- throughput screening (HTS)

To establish a reporter cell line suitable for high-throughput screening

(HTS) we generated HEK293T cells with stably integrated pcDNA-Firefly^R - ⁱⁿⁱ/Renilla^lni reporter plasmid. Single clones were tested for renilla and firefly expression and the highly expressing cell clones were in addition tested for stability of expression over repeated cell passages. Cell line clones that met these criteria were selected for further experiments. Similarly, a cell line with stably integrated pcDNA-Firefly^Rei/Renilla^Rei plasmid was generated. To determine an optimal assay window using reference

compounds we performed kinetic experiments with the mTORCl inhibitors rapamycin and PP242 over 24 hours. Both drugs showed the strongest decrease in TRI value at 8 hours, which was chosen as a suitable time point to perform the HTS (Fig EV2A).

We screened a library consisting of 780 FDA-approved drugs (ENZO #BML-2841) using the Firefly^R<! ""/Renilla^Im HEK293T reporter cell line. 1.5xl0 cells per well (50 μΐ volume) were seeded in a 384-well format and the Firefly^Re'^im/Renilla^Ini system was exposed to 10 μΜ of a single drug per well for 8h (three assay plates in total). Each assay plate contained 6 wells with 0.5% v/v DMSO added as reference (Fig 3A-C; bar graph at the right). Rapamycin was included as positive control on each plate (n=6) and induced a significant decrease in re-initiation/initiation ratio (Firefly^Re-ⁱⁿⁱ/¾enilla^lni) for each plate (plate 1, 0.82 ± 0.04; plate 2, 0.85 ± 0.025; plate 3, 0.9 ± 0.028) (Fig 3A-C; bar graph at the right). 45 drugs were found to decrease the translation re-initiation/initiation ratio (Firefly^{Re in}VReniIla^Tni) below the threshold of three times the standard deviation from the mean value of DMSO treated cells (hit rate 5.7%) (Fig 3A-C). The ENZO library contains three mTORCl inhibitors that are derived from ra amycin (the rapalogs everolimus, sirolimus and temsirolimus) and two of them, sirolimus and everolimus, decreased re-initiation/initiation ratio (Fig 3A, B). The temsirolimus signal was neglected because of aberrantly low luciferase signals (technical failure).

EXAMPLE 4: Validation of the drugs that decrease translation reinitiation/initiation ratio

Four drugs with the strongest effect on decreasing re-initiation/initiation ratio were chosen for further validation using counter assays (Fig 3A-C, numbered values #1-4)

clrug #l Raloxifene

drug #2 Febuxostat

drug #3 Thioguanine-6

drug #4 Adefovir Dipivoxil

See also Fig 3D for drug names and structural formulas. Treatment of the pcDNA-Firefly^{R f}/Renilla^}M or pcDNA3-Firefly^{Re ini}/Remlla^Ini containing HEK293T cell line using 10 μΜ of each drug for 8 h in a 96-well format revealed for drug #3 and #4 enhancement of the renilla reference signal compared to DMSO control (Fig EV2B, C). The calculated TRIs (see formula Fig IB) revealed that similar to the result of the HTS screen drug #4 showed the strongest effect compared to drug #2 and #3, however, we could not confirm an effect of drug #1 on the TRI (Fig 4A). Only drug #4 significantly lowered TRI values in two additional different cell lines, the mouse hepatoma cell line Hepal-6 and the human breast cancer cell hne MCF-7 (Fig 4B, C). Drugs #2 did significantly lower the TRI in Hepal-6 but not in MCF7 cells, and drug #3 did not alter the TRI in Hepal-6 and even increased the TRI in MCF7 cells (Fig 4B, C). These two drugs were excluded from further examination. To examine dose-dependency of treatment the TRI was measured for serially diluted concentrations between 1 nM and 100 μΜ for drugs #4 compared to drug #1 (Fig EV2D). Drug #4 showed a dose- response with a half maximal effective concentration (EC 50) of 1 μΜ, while drug #1 showed a much higher EC50 of 70.4 μΜ. Cytotoxicity over 8 hours treatment was examined using fluorescent cell viability assay (CellTiter- Fluor, Promega) and revealed for both drugs #1 and #4 only serious cytotoxicity at 100 μΜ concentrations (Fig EV2E). In addition, treatment with drug #4 lowered endogenous LIP/LAP ratio comparable to treatment with rapamycin in HEK293T, Hepa 1-6 and MCF-7 cells (Fig 4D-F). Taken together, the counter assays revealed that drug #4 (Adefovir Dipivoxil) is best in affect re-initiation measured as reduced TRI and lower LIP levels in cells that originate from three different tissues.

We have recently shown that suppression of LIP expression results in a metabolic shift towards more fatty acid oxidation in cell culture

experiments and using a mouse model that is deficient in LIP expression (C/EBPp^Au0RF mice) (Ziclek et al, 2015). To examine an eventual similar effect of drug #4 (Adefovir Dipivoxil) that also lowers LIP levels we measured fatty acid oxidation (FAO) of exogenously added palmitate-BSA in Hepal-6 cells using the Seahorse XF extracellular flux analyzer.

Surprisingly, treatment with Adefovir Dipivoxil resulted in an enhanced FAO-related oxygen consumption rate (OCR) that could be reverted by ectopic expression of LIP (Fig 5).

Taken together, our experiments show that the TRI reporter system can be successfully applied in HTS campaigns to identify compounds that alter the ratio between translation initiation and re-initiation depending on the C/EBPp-uORF-5'UTR, which potentially display CR-mimetic properties. REFERENCES

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Claims

1. A dual reporter plasmid system comprising a first nucleic acid sequence encoding a Renilla luciferase transcript and a second nucleic acid sequence encoding a firefly luciferase transcript, wherein said first and second nucleic acid sequence are both under control of the C/EBPp-5'UTR- uORF (upstream Open Reading Frame).

2. A cell comprising a dual reporter plasmid system according to claim 1.

3. Cell according to claim 2, wherein said plasmid is stably integrated into the genome of said cell.

4. A method for identifying a compound capable of mimicking calorie restriction comprising steps of

- providing a cell culture comprising cells according to claim 2 or 3;

- contacting said cell culture with a candidate compound to allow for luciferase expression;

determining expression of Renilla luciferase and firefly luciferase;

- calculating the translation re-initiation index (TRI) based on the

formula.. TRI = Firefly^{Re mitia, ,on} x Renilla^Reference / Renilla^Tniiia(io x

Firefly ^Iteferenc ; and

- correlating said TRI with the capacity of said compound to mimic calorie restriction, wherein a reduction in TRI is a positive indicator.

5. Method according to claim 4, further comprising the use of a reference cell comprising a reference dual reporter plasmid system

comprising a first nucleic acid sequence encoding a Renilla luciferase transcript and a second nucleic acid sequence encoding a firefly luciferase transcript, that are not under the control of C/EBPp-5'UTR-uORF

sequences.

6. Kit-of-parts comprising a dual reporter plasmid system according to claim 1, a luciferase substrate, and optionally a reference reporter plasmid.

7. A high throughput screening assay comprising the use of a method according to claim 4 or 5 and/or a kit-of-parts according to claim 6.

8. Adefovir dipivoxil for use in a method for improving the metabolic health in a subject.

9. Adefovir dipivoxil for use according to claim 8, wherein improving the metabolic health comprises shifting the metabolism towards enhanced beta-oxidation.

10. Adefovir dipivoxil for use according to claim 8, wherein improving the metabolic health comprises enhancing or supporting weight loss in said subject.

11. Adefovir dipivoxil for use according to any one of claims 8-10, wherein the subject is a human subject.

12. Adefovir dipivoxil for use according to any one of claims 8-11, wherein the subject is suffering from, or at risk of developing, a condition selected from the group consisting of metabolic syndrome, obesity, type 2 diabetes, pre-diabetes, hypertension, disorders of lipid metabolism, insuhn resistance, endothelial dysfunction, pro-inflammatory state, and pro- coagulative state.

13. Adefovir dipivoxil for use as a calorie restriction mimetic.

14. A weight management composition comprising Adefovir dipivoxilin combination with a further active ingredient supporting enhancing weight loss.