Multivalent MMP-12 inhibitors as a valuable approach to counteract the intestinal epithelial barrier impairment and inflammation in an in vitro model of obesity

doi:10.21203/rs.3.rs-4866971/v1

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Multivalent MMP-12 inhibitors as a valuable approach to counteract the intestinal epithelial barrier impairment and inflammation in an in vitro model of obesity

https://doi.org/10.21203/rs.3.rs-4866971/v1

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Intestinal epithelial barrier (IEB) impairment represents a prodromal event underlying obesity and related systemic inflammation. In this context, metalloproteinase-12 (MMP-12) has been reported to increase the IEB permeability through the reduction of tight junction protein expression. Herein we report our effort to develop a small series of MMP-12 inhibitors as potential agents able to counteract the IEB alterations and intestinal inflammation associated with obesity. Three multivalent and gut-restricted carboxylate-based selective inhibitors of MMP-12 were synthesized and tested first on human recombinant MMP-12 isolated enzyme and then on human intestinal epithelial Caco-2 cells treated with palmitate (PA) and lipopolysaccharide (LPS), to mimic the in vivo exposure to hypercaloric diet. Trimeric derivative 2 in particular showed a nanomolar activity against MMP-12 and was able to increase both ZO-1 and claudin-1 tight junction expression in a concentration-dependent manner, already at a concentration of 50 nM. This compound was also the most effective in reducing interleukin-1β release from Caco-2 cells treated with PA and LPS. This preliminary work indicates that a pharmacological modulation of MMP-12 represents a promising strategy to counteract the impairment of IEB integrity and intestinal inflammation associated with obesity.

MMP-12 inhibitor

biphenylsulfonamide

matrix metalloproteinases

multivalent effect

intestinal permeability

tight junction

Caco-2 cells

obesity

Obesity is a complex and multifactorial disease strongly linked to several medical comorbidities, such as type-2 diabetes mellitus, cardiovascular diseases and gastrointestinal disorders (i.e., infrequent bowel movements and constipation), which negatively impact the patients’ quality of life and complicate their clinical management [1, 2]. In this context, alterations of intestinal epithelial barrier (IEB) have been identified as primum movens of a series of pathophysiologic events underlying obesity and related disorders [3, 4]. Indeed, the excessive intake of foods with high energy density has been demonstrated to induce alteration of IEB integrity and permeability, thus facilitating the translocation of immunogenic products and pathogens into the mucosa and the bloodstream with consequent development of a condition of low-grade systemic inflammation, that affects the physiological functions of intestine and other organs [3, 4]. Based on this evidence, the identification of novel molecules able to counteract the impairment of IEB associated with obesity could represents a promising therapeutic strategy.

The human matrix metalloproteinase (MMP) family consists of 23 zinc-dependent endopeptidases responsible for extracellular matrix (ECM) degradation during wound healing and tissue remodeling [5, 6]. Among these, matrix metalloproteinase 12 (MMP-12) or macrophage metalloelastase is a well studied enzyme [7] which is upregulated in chronic obstructive pulmonary disease (COPD) [8], emphysema [9], atherosclerosis and inflammatory diseases [10]. This enzyme is first secreted in the extracellular space and, after activation of the pro-enzyme, the resulting catalytic domain can stay in the extracellular space or localize near the cell surface [11]. It’s role in important diseases, such as pulmonary or cardiovascular diseases, has promted the development of selective inhibitors as potential therapeutic agents [12–14] and of fluorescent probes or radiotracers for diagnostic purposes [15, 16].

Recently, MMP-12 has been reported to contribute to the degradation of tight junction proteins (TJs) and inflammatory processes in several pathological conditions, including obesity [17, 18]. Indeed, an increased expression of MMP-12 along with a reduction in the expression of TJ zonulin-1 and occludin has been reported in small intestinal tissues from obese mice fed with high fat-diet (HFD) [19], thus suggesting a correlation between the expression levels of MMP-12 and the reduction of intestinal TJs. In support to this view, Song et al. observed that MMP-12 deficiency induced by mi-RNA was able to alleviate the reduction of TJs and to reduce the intestinal inflammation associated with HFD [19], thus highlighting that the pharmacological modulation of MMP-12 could represents a promising strategy to restore the IEB permeability and to curb gut inflammation in such pathological condition.

Based on these findings, and considering the limitations deriving from the clinical use of mi-RNA, in the present study we report the synthesis of multivalent MMP-12 inhibitors potentially able to correct the small intestinal homeostasis as a new approach to treat obesity. These compounds were designed to be intestine-targeted due to a low intestinal absorption. Such a behaviour could allow to increase the local concentration of compound in the site of action and also to decrease the side effects due to a systemic inhibition of MMPs. The multivalent derivatives were first screened on isolated enzymes (MMP-12 and other MMPs) to prove their inhibitory activity towards the target enzyme, then in an in vitro model of obesity.

Desing

To develop gut-restricted agents, the chemical structure of a carboxylate-based MMP-12 inhibitor previously developed by our group, EN238,[20,21] was modified to give the multivalent derivatives 1-3 (Figure 1). Three features widely reported in literature [22] to obtain non-absorbable small-molecule drugs were exploited to design the new compounds: a high molecular weight, incorporation of a positively charged moiety and incorporation of highly polar polyethylene glycol (PEG) side chains. All these approaches, separately or jointly applied, should limit intestinal absorption of orally administered compounds due to a low passive diffusion through intestinal epithelium.

Compound EN238 was chosen as starting point due to its nanomolar inhibitory activity against MMP-12 (IC₅₀= 35 nM), the presence of a carboxylate as a weak chelating function which should guarantee a lower toxicity and higher stability with respect to the most used hydroxamate [23], and a low intestinal permeability as already reported [21]. In a previous study aiming to develop MMP-9 inhibitors able to impair natural MMP-9 homodimerization, compound 1 was obtained by symmetrical dimerization of EN238, exploiting the isophthalamide nucleus as central core [24]. This bivalent inhibitor proved to reduce invasion in three glioma cell lines and to selectively inhibit the homotrimeric MMP-9 proteoform [25], moreover the crystallographic structure of the complex between 1 and MMP-12 was solved (PDB ID: 4H30) [26]. In the present work, derivative 2 was designed to have a trimeric structure given by the repetition of the matrix metalloproteinase inhibitor (MMPI) scaffold (see Scheme 2) linked to a benzene central ring through a PEGylated chain. Finally, compound 3 derived from 1 after introduction of a charged and PEGylated side chain in 5 position on the central ring. According to this strategy, all three derivatives contained the same pharmacophore originally present in EN238, the biphenylsulfonamido-based carboxylate indicated in red in Figure 1 and responsible for inhibitory activity against MMP-12, that was differently tethered to a so called “kinetophore” [27], a polar or charged moiety which has influence on the pharmacokinetic profile, at a position non-essential for activity. Moreover, the use of multivalent derivatives bearing two or three warheads could improve pharmacological activity of MMP inhibitors as already shown for other classes of drugs [28,29].

Chemistry

Compound 1 has been synthesized as previously reported by Nuti et al. [24]. The new inhibitors 2 and 3 have been synthesized as described in Scheme 1 and 2, respectively. The crucial step of all synthetic pathways here reported is the condensation promoted by the system carbodiimide-based 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-Hydroxybenzotriazole (HOBt), fundamental in yielding strategic amide bonds. Amide bonds are typically obtained by reacting an activated carboxylic acid with an amine [30]. Various protocols and reagents have been developed over time to activate carboxylic acids, such as the systems DCC/DMAP, EDC/HOBt, HATU/DIPEA, CDI/Et₃N, and BOPCl/Et₃N [31]. However, when electron-deficient systems such as aromatic amines are involved, amide coupling tends to be sluggish and yields less satisfactory results. In this work we adopted the EDC/HOBt coupling strategy for different steps of our synthesis obtaining all amide bonds with yield over 50% even with aromatic amines and contemporary multiple conjugations. EDC efficiently mediates conjugation reaction and the support of HOBt is significative in order to reduce epimerization, prevent side reactions, and therefore increase reaction efficacy [32]. Moreover, both reagents form water-soluble side-products that can be easily separated from the reaction crude.

The synthesis of compound 2 is reported in Scheme 1. The trifluoroacetate amine 4, synthesized as already described [20,21], was conjugated with Boc-NH-(PEG)₃-COOH 5 using EDC/HOBt system followed by an acid hydrolysis with trifluoroacetic acid (TFA) in controlled conditions (1h at 0°C) in order to remove the Boc protection without affecting the tert-butyl group, to give the trifluoroacetate amine 7. The crucial condensation among three units of 7 and the central scaffold represented by benzene-1,3,5-tricarboxylic acid 8 was stoichiometrically (3:1) achieved using the same condensation system, followed by purification through reverse phase liquid chromatography. The final acid hydrolysis of intermediate 9 in standard conditions yielded the target trimeric derivative 2 after the contemporary removal of the three tert-butyl ester groups.

The PEGylated dimeric inhibitor 3 was synthesized as reported in Scheme 2. PEGylated dimethyl isophthalate 11 was obtained by condensation between the commercial dimethyl 5-aminoisophthalate 10 and the PEGylated derivative 5 using the conjugation system EDC/HOBt. The amine 12 was obtained as a trifluoroacetate salt by acid hydrolysis of the Boc group upon treatment of compound 11 with TFA. These two steps were repeated in order to elongate the hydrophilic chain by adding again the PEG 5 followed by TFA-mediated hydrolysis, to finally give derivative 13. The methyl ester functions of compound 13 were hydrolyzed under basic conditions by treatment with LiOH 1M solution to afford the dicarboxylic acid 14. The conjugation between dicarboxylic acid 14 and 2 equivalents of the MMPI amine 4, was obtained by EDC/HOBt system, followed by acid hydrolysis useful to remove the tert-butyl and the Boc protection in one step thus affording the target PEGylated-dicarboxylic acid 3.

Enzyme inhibition assays

The newly synthesized multivalent compounds 2 and 3 were tested on human recombinant MMP-12 by a fluorometric assay to prove their activity in comparison with the parent compound EN238 and its bivalent derivative 1. The inhibitory activity of the four carboxylate-based compounds was evaluated also against MMP-1, -2 and -9 to verify the selectivity over other metalloproteases belonging to the same family. MMP-1 in particular is considered an anti-target MMP responsible for musculoskeletal side-effects typical of broad spectrum MMP inhibitors (MMPIs) [33]. In fact, among the principal obstacles that have hindered clinical development of MMPIs for cancer or other therapies so far there is an inadequate selectivity for the target enzyme with consequent long-term toxicity that has caused a forced interruption of therapy during several clinical trials [34,35].

Table 1. In vitro enzymatic activity^a (IC₅₀, nM) of compounds used in the study.

Compd	IC₅₀ (nM)
Compd	MMP-1	MMP-2	MMP-9	MMP-12
EN238 [20]	16000±840	170±13	510±44	35±2.3
1 [24]	11800±740	63±3.5	26±2.7	1.3±0.1
2	6540±1740	27±2.7	104±2.8	3.0±0.36
3	4135±690	18±1.7	113±1.95	1.8±0.08

^aAssays were run in triplicate. The final values given here are the mean ± SEM of three independent experiments.

As can be seen from Table 1, all multivalent derivatives showed a nanomolar inhibitory activity against MMP-12 with a 10 to 27-fold increase with respect to the reference compound, EN238. These results indicate that all structural modifications to EN238 did not substantially alter the affinity for the target enzyme, MMP-12. On the contrary, as hypothesized, the multivalency of new compounds seems to have a positive effect on activity that should be further investigated. Moreover, all three compounds improved the selectivity over MMP-1 and MMP-9 with respect to EN238, and maintained the selectivity over MMP-2. Overall, the bivalent derivative 1 showed the best results of both activity (IC₅₀= 1.3 nM) and selectivity for MMP-12.

At this point, the octanol/water partition coefficient (cLogP) of the three multivalent derivatives 1-3 were calculated in silico in comparison with EN238 by using MolBook UNIPI Software [36] (Table 2) to predict their ability to be absorbed from the intestine by passive diffusion. According to Lipinski’s Rule of 5 [37] compounds with a molecular weight (MW) greater than 500 Da, and a clogP greater than 5 should guarantee a very low systemic bioavailability. Also molecules that have topological polar surface area (TPSA) > 140 Å² are less likely to reach systemic circulation [38]. On the basis of these calculations, EN238 derivatives 1 and 2 showed a higher clogP than the parent compound with trimeric derivative 2 having the lowest probability to be absorbed by intestinal epithelial cells, clogP = 7, TPSA = 509 and MW= 2027. Only derivative 3 presented a clogP value comparable to that of the parent compound.

Table 2. Calculated logP values and molecular weight (MW) of 1-3 and reference compound EN238.

Compound	cLogP^a	TPSA^a	MW
1	6.08	207.56	883.04
2	7	509.4	2027.37
3	3.59	407.37	1506.65
EN238	3.88	103.78	480.58

^aCalculated using MolBook UNIPI (Beta Release 1.5).

Cell-based assays

A set of in vitro experiments were performed to evaluate the efficacy of the multivalent MMP-12 inhibitors in counteracting the IEB alterations and intestinal inflammation associated with obesity. To purpose this aim, human intestinal epithelial Caco2 cells were treated with palmitate (PA, the major source of HFD) and lipopolysaccharide (LPS, index of endotoxemia associated with obesity) to mimic the in vivo features of a hypercaloric diet exposure.

Expression of tight junction proteins

Treatment of Caco2 cells with LPS and PA induced a significant decrease in zonulin-1 (ZO-1), and claudin-1 expression (Figure 2A, 2B), indicating that a condition mimicking HFD exposure induces alterations in IEB integrity [39]. Of interest, treatment with 1 and 2 at a concentration of 50 and 100 nM was able to counteract significantly the deleterious effect of LPS and PA on claudin-1 expression in a dose-dependent way (Figure 2B) but only trimeric derivative 2 was able to increase ZO-1 expression in a dose-dependent way (Figure 2A). By contrast, incubation with 3 was able to prevent the reduction of claudin-1 but not of ZO-1 (Figure 2A, 2B). Overall, these results suggest that the selective inhibition of MMP-12 could represent a valuable tool to counteract the impairment of IEB integrity in the context of obesity.

IL-1β release

Several studies have reported that at sites of inflammation the active form of interleukin-1β (IL‐1β) and MMPs, including MMP-12, are simultaneously expressed [40]. In particular, IL-1β can influence the expression and activation of MMPs, and viceversa, thus indicating the existence of a reciprocal interaction. In this context, IL‐1β plays an important role in modulating the expression of intestinal TJs and consequently the IEB integrity [41,42]. In particular, a massive IL‐1β release contributes to induce a reduction in TJ expression with consequently increase in gut permeability. Accordingly, we decided to investigate the effects of our MMP-12 inhibitors in counteracting the IL‐1β release from Caco2 cells under condition mimicking the obesity status. Treatment of Caco-2 cells with LPS and PA significantly increased the release of IL-1β, as compared with control cells (Ctrl) (Figure 2C). Such an increase was reduced by treatment with 1, 2 and 3 at higher dose, thus highlighting an anti-inflammatory activity of these compounds. Also in this assay, compound 2 showed the best results, with a reduction of IL-1β release of 19% at 100 nM.

Experimental part

Chemistry

Melting points were determined on a Koﬂer hotstage apparatus and are uncorrected. ¹H, ¹³C and ¹⁹F NMR spectra were recorded on a Bruker Avance III HD 400 MHz spectrometer using the indicated deuterated solvents. Chemical shifts (δ) are reported in parts per million and coupling constants (J) are reported in hertz (Hz). ¹³C NMR spectra were fully decoupled. The following abbreviations were used to explain multiplicities: singlet (s), doublet (d), triplet (t), double doublet (dd), broad (br), and multiplet (m). Chromatographic separations were performed on silica gel columns by ﬂash column chromatography (Kieselgel 40, 0.040−0.063 mm, Merck) or using ISOLUTE Flash Si II cartridges (Biotage). Reactions were followed by thin-layer chromatography (TLC) on Merck aluminum silica gel (60 F254) sheets that were visualized under a UV lamp. Evaporation was performed in vacuo (rotating evaporator). Sodium sulfate was always used as the drying agent. Commercially available chemicals were purchased from Sigma-Aldrich (Merck). The ≥ 95% purity of the tested compounds was confirmed by combustion analysis. Analytical results are within ± 0.4% of the theoretical values. The ESI-MS spectra were recorded by direct injection at 5 (positive) and 7 (negative) μl min−1 flow rate in an Orbitrap high-resolution mass spectrometer (Thermo, San Jose, CA, USA), equipped with HESI source.

Synthetic details and characterization of compounds

General procedure for the condensation reaction using EDC/HOBt system for the synthesis of amides 6, 9, 11, 13, 15

To a solution of the appropriate carboxylic acid (2.25 eq) in dry DCM (5 mL/mmol), the appropriate amine (1 eq), Et₃N (2.25 eq), HOBt (2.25) and finally EDC (2.25) were added. The reaction mixture was stirred at room temperature for 12h, under argon atmosphere. Then the solvent was evaporated and EtOAc was added and washed with a saturated solution of NaHCO₃, HCl 1N and brine. The organic phase was dried over Na₂SO₄, filtered and evaporated under reduced pressure.

(S)-24-([1,1'-biphenyl]-4-ylsulfonyl)-25-isopropyl-2,2-dimethyl-4,20-dioxo-3,8,11,14,17-pentaoxa-5,21,24-triazahexacosan-26-oic acid (6). The title compound was synthesized from amine 4 and carboxylic acid 5 following the general procedure. The pure compound was obtained as a colorless oil (97% yield). ¹H NMR (400 MHz, CDCl₃) δ: 7.90-7.88 (m, 2H), 7.69-7.66 (m, 2H), 7.57-7.55 (m, 2H), 7.49-7.41 (m, 3H), 6.99 (bs, 1H), 5.11 (bs, 1H), 4.11 (d, J= 7.2 Hz, 1H), 3.96-3.83 (m, 1H), 3.75 (t, J= 5.6 Hz, 2H), 3.66-3.47 (m, 12H), 3.38-3.29 (m, 4H), 3.19-3.12 (m, 2H), 2.83-2.81 (m, 2H), 2.49 (t, J= 6.0 Hz, 2H), 2.05-2.03 (m, 1H), 1.43 (s, 9H), 1.23 (s, 9H), 1.05, 0.95 (2xd, J= 6.4 Hz, each 3H).

Trimeric derivative 9: The title compound was prepared from amine 7 (130 mg, 0.16 mmol, 4 eq) and the commercial benzene-1,3,5-tricarboxylic acid 8 (8.6 mg, 0.041 mmol, 1 eq) in dry DMF (5mL). Et₃N (0.15 mL, 1.11 mmol, 27 eq), HOBt (55 mg, 0.41 mmol, 10 eq), EDC (78.50 mg, 0.41 mmol, 10 eq) were added following the general procedure. The crude residue was purified by reverse phase flash chromatography using a C-18 Isolute cartridge (SI -C18 5g) (H₂O/MeOH 1:2) affording the pure compound 9 as a colorless oil (47 mg, 52% yield). ¹H NMR (400 MHz, CDCl₃) δ: 8.54 (s, 3H), 8.05 (bs, 3H), 7.90-7.87 (m, 6H), 7.68-7.66 (m, 6H), 7.56-7.54 (m, 6H), 7.47-7.40 (m, 9H), 7.11 (bt, 3H), 3.92 (d, J= 7.2 Hz, 3H), 3.81-3.76 (m, 4H), 3.68-3.47 (m, 57H), 3.36-3.34 (m, 3H), 2.39 (t, J= 6.0 Hz, 6H), 2.16-2.11 (m, 3H), 1.21 (s, 27H), 1.02, 0.93 (2xd, J= 6.4 Hz, each 9H).¹³C NMR (100 MHz, CDCl₃) δ:171.9, 169.7, 166.6, 145.7, 139.3, 138.2, 135.1, 129.1, 129.9, 128.5, 128.1, 127.7, 127.3, 82.1, 70.4, 70.2, 70.1, 70.0, 69.9, 67.0, 66.6, 44.1, 40.3, 40.0, 36.6, 29.2, 27.7, 20.1, 19.3.

Dimethyl 5-(2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-amido)isophthalate (11): Amide 11 was synthesized from the commercial Boc-NH-(PEG)₃-COOH 5 and dimethyl 5-aminoisophthalate 10 following the general procedure. The crude was purified by flash chromatography using an Isolute cartridge (SI 10g) (100% CHCl₃) affording the pure compound as a colorless oil (53% yield). ¹H NMR (400 MHz, CDCl₃) δ: 9.14 (bs, 1H), 8.44-8.43 (m, 2H), 8.40-8.39 (m, 1H), 3.92 (s, 6H), 3.84 (t, J= 5.12 Hz, 2H), 3.71 (s, 6H), 3.67-3.61 (m, 2H), 3.57-3.55 (m, 4H), 3.52 (t, J= 5.12 Hz, 2H), 3.28 (t, J= 5.12 Hz, 2H) 2.69 (t, J= 5.12 Hz, 2H), 1.43 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ: 170.6; 166.2; 156.2; 139.2; 131.3; 126.1; 125.2; 70.8; 70.6; 70.5; 70.4; 70.3; 67.1; 52.5; 40.5; 38.0; 28.6.

dimethyl 5-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17,24,27,30,33-nonaoxa-5,21-diazahexatriacontan-36-amido)isophthalate (13). The title compound was synthesized from amine 12 and Boc-NH-(PEG)₃-COOH 5 following the general procedure. The pure compound was obtained as a colorless oil (100% yield). ¹H NMR (400 MHz, CDCl₃) δ: 9.17 (bs, 1H), 8.46-8.44 (m, 2H, Ar), 8.40-8.39 (m, 1H), 6.81 (bs, 1H), 5.07 (bs, 1H), 3.92 (s, 6H), 3.84 (t, J= 5.6 Hz, 2H), 3.71 (s, 6H), 3.65-3.67 (m, 21H), 3.54-3.42 (m, 4H), 3.42- 3.41 (m, 2H), 3.31-3.28 (m, 2H), 2.69 (t, J= 6 Hz, 2H), 2.48 (t, J= 6 Hz, 2H), 1.43 (s, 9H).

di-tert-butyl 2,2'-((((5-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17,24,27,30,33-nonaoxa-5,21-diazahexatriacontan-36-amido)isophthaloyl)bis(azanediyl))bis(ethane-2,1-diyl))bis(([1,1'-biphenyl]-4-ylsulfonyl)azanediyl))(2S,2'S)-bis(3-methylbutanoate)(15). Amide 15 was synthesized from amine 4 (92 mg, 0.1680 mmol, 2 eq) and dicarboxylic acid 14 (64 mg, 0.084 mmol, 1 eq) in dry DMF (2 mL) following the general procedure. The crude was purified by flash chromatography using an Isolute cartridge (SI 5g) (in gradient from 100% CHCl₃to CHCl₃:MeOH 50:1) affording the pure compound as a colorless oil (66 mg, 46% yield).¹H NMR (400 MHz, CDCl₃) δ: 9.08 (s, 1H), 8.22 (s, 2H), 8.02 (bs, 1H), 7.93-7.91 (m, 4H), 7.55-7.53 (m, 4H), 7.47-7.42 (m, 8H), 6.99 (bs, 1H), 3.93-3.92 (m, 4H), 3.84 (t, J= 5.6 Hz, 2H), 3.77-3.61 (m, 40H), 3.33-3.27 (m, 4H), 2.67 (t, J= 5.2 Hz, 2H), 2.49 (t, J= 6 Hz, 2H), 1.42 (s, 9H), 1.24 (s, 18H), 1.05, 0.89 (2xd, J= 6.4 Hz, each 6H).

General procedure for the TFA-mediated acid hydrolysis for the synthesis of compounds 7, 2, 12, 3

To a solution of the appropriate NH-Boc or tBu protected derivative (1 eq) in DCM (20 mL/mmol), TFA (30-342 eq) was added dropwise at 0°C. After completion of the reaction monitored by TLC, the solvent was co-evaporated with toluene (3x) and Et₂O (3x).

(S)-19-([1,1'-biphenyl]-4-ylsulfonyl)-20-carboxy-21-methyl-15-oxo-3,6,9,12-tetraoxa-16,19-diazadocosan-1-amine trifluoroacetate salt (7). The title compound was prepared from NH-Boc-protected derivative 6 (180 mg, 0.235 mmol) treated dropwise with TFA (0.5 mL, 30 eq). The reaction was stirred for 1h at 0°C and then treated following the general procedure. The crude residue was purified by flash chromatography using an Isolute cartridge (SI 10g) (CHCl₃:MeOH 50:1) to give compound 7 as a colorless oil (130 mg, 70% yield). ¹H NMR (400 MHz, CDCl₃) δ: 7.94-7.90 (m, 2H), 7.89-7.87 (m, 2H), 7.77 (bs, 1H), 7.71-7.68 (m, 2H), 7.58-7.56 (m, 2H), 7.52-7.46 (m, 3H), 7.43-7.39 (m, 1H), 3.77 (d, J= 6 Hz, 1H), 3.77-3.75 (m, 2H), 3.73-3.45 (m, 18H), 3.22 (bs, 2H), 2.65-2.64 (m, 2H), 2.13-2.07 (m, 1H), 1.23 (s, 9H), 1.02, 0.95 (2xd, J= 6.8 Hz, each 3H).¹⁹F NMR (376 MHz, CDCl₃) δ: -75.9.

Trimeric derivative 2. The title compound was prepared from tert-butyl ester 9 (47 mg, 0.021 mmol) treated dropwise with TFA (0.56 mL, 342 eq). The reaction was stirred for 15h at rt and then treated according to the general procedure to give final compound 2 as a white solid (45 mg, quantitative yield) after trituration with n-hexane. Mp: 70-73°C; ¹H NMR (400 MHz, acetone-d₆) δ: 8.49 (s, 3H), 7.95-7.93 (m, 6H), 7.84-7.82 (m, 6H), 7.72-7.70 (m, 6H), 7.52-7.48 (m, 6H), 7.44-7.41 (m, 3H), 4.07 (d, J= 6 Hz, 3H), 3.66-3.51 (m, 64H), 3.31-3.23 (m, 4H), 2.37 (t, J= 6 Hz, 6H), 2.23-2.12 (m, 3H), 1.02, 0.97 (2xd, J= 6.8 Hz, each 9H).¹³C NMR (100 MHz, acetone-d₆) δ: 172.0, 166.6, 145.8, 139.7, 138.9, 135.9, 129.8, 129.4, 129.2, 128.9, 128.0, 127.9, 71.0, 70.9, 70.8, 70.6, 70.2, 67.6, 66.6, 44.8, 40.5, 40.4, 37.1, 20.3, 19.6. HRMS (ESI, m/z) calculated for C₉₉H₁₃₅N₉O₃₀S₃ [M−H]⁻: 2024.84042; found: 2024.83972. Elemental analysis calcd (%) for C₉₉H₁₃₅N₉O₃₀S₃ : C 58.65, H 6.71, N 6.22; found: C 58.71, H 6.75, N 6.25.

15-((3,5-bis(methoxycarbonyl)phenyl)amino)-15-oxo-3,6,9,12-tetraoxapentadecan-1-amine trifluoroacetate salt (12): The title compound was prepared from NH-Boc-protected compound 11 (320 mg, 0.5608 mmol) treated dropwise with TFA (1.3 mL, 30 eq). The reaction mixture was stirred for 3h at 0°C and then treated following the general procedure to give the trifluoroacetate salt amine 12 as a white solid. ¹H NMR (400 MHz, CDCl₃) δ: 9.38 (bs, 1H), 8.44-8.43 (m, 2H, Ar), 8.41-8.40 (m, 1H, Ar), 7.53 (bs, 3H), 3.93 (s, 6H), 3.88 (t, J= 5.6 Hz, 2H), 3.78-3.76 (m, 2H) 3.69-3.59 (m, 12H), 3.17-3.13 (m, 2H) 2.80 (t, J= 5.6 Hz, 2H). ¹⁹F NMR (376 MHz, CDCl₃) δ: -75.9.

(2S,2'S)-2,2'-((((5-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17,24,27,30,33-nonaoxa-5,21-diazahexatriacontan-36-amido)isophthaloyl)bis(azanediyl))bis(ethane-2,1-diyl))bis(([1,1'-biphenyl]-4-ylsulfonyl)azanediyl))bis(3-methylbutanoic acid)(3): The title compound was prepared from compound 15 (75 mg, 0.047 mmol) treated dropwise with TFA (0.52 mL, 144 eq). The reaction was stirred for 5h at rt and then treated following the general procedure to give the trifluoroacetate salt amine 3 (60 mg, 83%yield) as a white semisolid after trituration with Et₂O. ¹H NMR (400 MHz, CD₃OD) δ: 8.17 (d, J= 1.2 Hz, 1H), 7.98-7.94 (m, 4H, Ar), 7.96-7.94 (m, 4H, Ar), 7.64-7.62 (m, 4H), 7.47-7.38 (m, 7H), 4.09 (d, J= 10.4 Hz, 2H), 3.93-3.46 (m, 48H), 3.11 (t, J= 5.6 Hz, 2H), 2.66 (t, J= 5.6 Hz, 2H), 2.45-2.42 (m, 2H), 1.06, 1.02(2xd, J= 6.4 Hz, each 6H); ¹³C NMR (100 MHz, CD₃OD) δ:172.8, 172.7, 171.2, 167.5, 145.5, 139.1, 137.9, 135.1, 128.7, 127.9, 127.1, 126.8, 121.5, 120.9, 70.1, 70.0, 69.9, 69.8, 69.7, 69.4, 66.8, 66.4, 43.5, 40.2, 39.1, 38.9, 37.2, 35.9, 28.5, 19.3, 18.5. ¹⁹F NMR (376 MHz, CDCl₃) δ: -76.9. HRMS (ESI, m/z) calculated for C₆₈H₉₃N₇O₂₀S₂ free base [M−H]⁻: 1390.58440; found: 1390.58447. Elemental analysis calcd (%) for C₇₀H₉₄F₃N₇O₂₂S₂ : C 55.80, H 6.29, N 6.51; found: C 55.83, H 6.31, N 6.56.

Synthesis of 5-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17,24,27,30,33-nonaoxa-5,21-diazahexatriacontan-36-amido)isophthalic acid (14)

To a solution of the dimethyl ester 13 (213 mg, 0.27 mmol) in 6 mL of MeOH, a solution of LiOH 1N (2.6 mL) was added. The reaction was stirred for 3 h and then evaporated under reduced pressure. The crude was dissolved in water and the solution was acidified up to pH<2. The acid solution was extracted with EtOAc (3x) and the combined organic extracts were dried over Na₂SO₄, filtered and evaporated to give compound 14 as a white semisolid (165 mg, 80% yield). ¹H NMR (400 MHz, DMSO-d₆) δ: 13.15 (bs, 2H), 10.30 (s, 1H), 8.44-8.43 (m, 2H, Ar), 8.14-8.13 (m, 1H), 7.87 (m, 1H), 6.71 (bs, 1H), 3.71 (t, J= 5.6 Hz, 2H), 3.57-3.36 (m, 30H), 3.17-3.15 (m, 2H), 2.30 (m, t J= 5.6 Hz, 2H), 1.36 (s, 9H)

Biological assays

Enzyme inhibition assays.

Human proMMP-9 was produced by recombinant expression in Sf9 insect cells and purified by gelatin-Sepharose chromatography. Next, proMMP-9 was activated by incubation with the catalytic domain of MMP-3 (cat. No. 444217, Merck Millipore, Darmstadt, Germany) as previously described [43]. Recombinant human proMMP-1 and proMMP-2 were purchased from R & D systems (Minneapolis, MN, USA), dissolved in assay buffer (150 mM NaCl, 5 mM CaCl2, 0.01% Tween-20, 50 mM Tris, pH 7.4) to a concentration of 100 µg/mL and activated by incubation with 1 mM p-aminophenylmercuric acetate for, respectively, 4h and 1h. To measure MMP-1, MMP-2 and MMP-9 inhibition, the activated MMPs were incubated with different concentrations of inhibitors and incubated for 30 min at 37 °C. Next, the OmniMMP substrate peptide (Mca-PLGL-Dpa-AR-NH₂, cat. no. BML-P126-0001, Enzo Life Sciences, Farmingdale, NY, USA) at a final concentration of 2.5 µg/mL was added and the increase in fluorescence over time was measured with the CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany). Proteolytic activity was determined by linear regression of the fluorescence curve and the percentage inhibition was calculated through comparison with the positive control (no inhibitors).

Recombinant human proMMP-12 was purchased from R&D Systems (Minneapolis, MN, USA). Pro-MMP-12 was auto activated by incubating in fluorometric assay buffer (FAB: Tris 50 mM, pH 7.5, NaCl 150 mM, CaCl₂ 10 mM, Brij-35 0.05%, and DMSO 1%) for 30 h at 37 °C. For assay measurements, the inhibitor stock solutions (DMSO, 10 mM) were further diluted in FAB. Activated enzyme (final concentration of 2.3 nM) and inhibitor solutions were incubated in the assay buffer for 3 h at 25 °C. After the addition of 200 μM solution of the fluorogenic substrate Mca-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH₂ (Bachem) in DMSO (final concentration of 2 μM), the hydrolysis was monitored every 10 s for 15 min, recording the increase in fluorescence (λ_ex = 325 nm, λ_em = 400 nm) with a Molecular Devices SpectraMax Gemini XPS plate reader (Molecular Devices, Sunnyvale, CA, USA). The assays were performed in triplicate in a total volume of 200 μL per well in 96-well microtiter plates (Corning black, NBS). Control wells lack inhibitor. The MMP inhibition activity was expressed in relative fluorescent units (RFU). Percent of inhibition was calculated from control reactions without the inhibitor. IC₅₀ was determined using the formula v_i/v₀ = 1/(1 + [I]/IC₅₀), where v_i is the initial velocity of substrate cleavage in the presence of the inhibitor at concentration [I] and v_o is the initial velocity in the absence of the inhibitor. Results were analyzed using SoftMax Pro software version 5.4.3 (Molecular Devices, Sunnyvale, CA, USA) and Prism Software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA).

Cell-based assays.

Cell culture

Human intestinal epithelial Caco-2 cell line were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 2 mM glutamine and 100 unit/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5 % CO₂. Caco-2 cells were kindly donated by Prof Guido Bocci (Department of clinical and Experimental Medicine, University of Pisa).

Cell treatments

Caco-2 cells were seeded at density of 5x10⁵ cells in 6-well plate and treated for 48h with lipopolysaccharide (LPS, 10 μg/mL, Sigma-Aldrich) and palmitate (PA, 400 µM, Sigma-Aldrich) to mimic the in vivo features of HFD exposure, in the absence or in the presence of MMP-12 inhibitors 1-3 (50 nM, 100 nM). Controls were run in parallel. Concentrations of PA and LPS were selected in accordance with previous reports [44–48]. Concentrations of compounds 1, 2 and 3 were selected based on IC₅₀ and dose-finding experiments were performed to select the two doses more effective in counteracting the reduction of TJ proteins and IL-1β levels. After 72h, Caco-2 cells were lysed and culture media were collected.

Western blot assays

Cells were lysed as previously described [49,50] and proteins were quantified using Bradford assay. Proteins were separated on a pre-cast 4–20% polyacrylamide gel (Mini-PROTEAN® TGX gel, Biorad) and transferred to PVDF membranes (Trans-Blot^® TurboTM, PVDF Transfer packs, Biorad). Then membranes were blocked with 3% BSA diluted in TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against GAPDH (#5174s, Cell Signaling), ZO-1 (Ab96587, Abcam) and claudin-1 (ab15098, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040 and anti-rabbit ab6721). Protein bands were detected with chemiluminescent ECL reagents (Clarityᵀᴹ Western ECL Blotting Substrate, Biorad). For the densitometry analysis, iBright Analysis software was used.

Assessment of interleukin (IL)-1β

The release of IL-1β from Caco2 cells was measured by ELISA kits (Abcam). Culture media were centrifuged for 5 min at 800 rpm to obtain cell-free supernatants. Supernatants (150 mL) were then used. Absorbance was expressed in percentage versus respective control.

Statistical analysis

The results are presented as mean ± S.E.M. of at least 3 independent experiments. The signiﬁcance of differences was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. P values <0.05 were considered signiﬁcantly different. All statistical procedures were performed by commercial software (GraphPad Prism, version 7.0 from GraphPad Software Inc., San Diego, CA, USA).

In conclusion, a recent study conducted by Song et al. [19] showed that HFD intake induced a marked increase in gut permeability through the reduction of TJ expression in obese mice, thus corroborating the deleterious effect of a hypercaloric diet consumption on IEB integrity. Of interest, in the same study, the authors observed that the MMP-12 deficiency was able to alleviate the reduction of TJs and to reduce the intestinal inflammation associated with HFD [19]. Based on this evidence, we decided to develop a small series of MMP-12 inhibitors as potential agents able to counteract the IEB alterations and intestinal inflammation associated with obesity. In this work, three multivalent and gut-restricted derivatives of the biphenylsulfonamido-based MMP-12 inhibitor EN238 were designed and synthesized to be evaluated in vitro. The kinetophore strategy was applied to modify EN238 physicochemical properties and reduce intestinal absorption without possibly altering affinity for the target enzyme. The three compounds, endowed with a high MW and highly polar PEGylated side chains, were first tested on some recombinant human MMPs to prove their activity and selctivity against the target enzyme and then on Caco-2 cells, treated with PA and LPS, representing an in vitro model of obesity. Enzymatic assays confirmed the inhibitory activity towards MMP-12 in the nanomolar range with small differences among derivatives and cell-based studies highlighted a strengthening effect on the intestinal epithelial barrier and an anti-inflammatory activity of our compounds. In particular, trimeric derivative 2 resulted the only one able to increase both claudin-1 and ZO-1 expression in a dose-dependent way, already at a concentration of 50 nM, and was the most effective in reducing IL-1β release from Caco-2 cells treated with PA and LPS. Of note, the anti-inflammatory and strengthening effects of trimeric derivative 2 can be ascribed, at least in part, to its ability in counteracting the massive release of IL-1β from intestinal epithelial cells through the inhibition of MMP-12. In addition, the efficacy of 2 can derive from a higher concentration in the site of action, on the luminal side of the gut where MMP-12 is secreted by epithelial cells, and/or from its higher multivalency with respect to the other compounds, 1 and 3. However, focused studies to investigate the molecular mechanisms underlying the trimeric derivative 2 activity are needed. Therefore, we are planning a set of in vivo experiments on obese mice to test the beneficial effects of this compound in preventing the IEB impairment and curbing the intestinal inflammation associated with obesity

LPS

lipopolysaccharide

palmitate

ZO-1

zonulin-1

Author contributions

D. C., V. D. G: investigation, methodology, validation, writing – original draft. C. M., C. D. S., L. B., J. V., L. A.: investigation, methodology, validation. A. R.: funding acquisition, project administration, supervision, writing – review & editing. M. M.: project administration, supervision, writing – review & editing. M. F., E. N.: conceptualization, methodology, validation, supervision, funding acquisition, writing – original draft. All authors have read and approved the published version of the manuscript.

Declaration of Competing Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments: This research was funded by University of Pisa (PRA_2022_19) and the “Fondi di Ateneo” 2021 to A.R. and E.N. We thank CISUP - Centre for Instrumentation Sharing - University of Pisa for the acquisition and elaboration of the HRMS spectra.

Supplementary information

Experimental detail,¹H-, ¹³C-NMR and HRMS spectra of final compounds. Dose-response curves for enzymatic inhibition of final compounds 2 and 3.

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Scheme 1 and 2 are available in the Supplementary Files section.

Onlinefloatimage2.png
Scheme 1. Reagent and conditions: i) EDC, HOBt, Et₃N, DCM, 12h, 97%; ii) TFA, DCM, 0°C, 1h, 70%; iii) EDC, HOBt, Et₃N, DCM, 12h, 52%; iv) TFA, DCM, rt, 15h, quantitative.
Onlinefloatimage3.png
Scheme 2. Reagent and conditions: i) EDC, HOBt, Et₃N, DCM, 12h, 53%; ii) TFA, DCM, rt, 3h, quantitative; iii) 5, EDC, HOBt, Et₃N, DCM, 12h, quantitative; iv) LiOH, MeOH, rt, 3h, 80%; v) EDC, HOBt, Et₃N, DCM, 12h, 46%; vi) TFA, DCM, rt, 5h, 83%.

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Editorial decision: Minor Revisions
04 Nov, 2024
Reviewers agreed at journal
16 Oct, 2024
Reviewers invited by journal
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Editor assigned by journal
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First submitted to journal
08 Aug, 2024

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Multivalent MMP-12 inhibitors as a valuable approach to counteract the intestinal epithelial barrier impairment and inflammation in an in vitro model of obesity

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Abstract

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Introduction

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EN238 [20]

1 [24]

2

3

Materials and methods

Conclusion

Abbreviations

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Supplementary Files

Status:

Version 1