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. 2021 Feb 24;12(3):477–484. doi: 10.1021/acsmedchemlett.1c00006

Discovery of a Lead Compound for Specific Inhibition of Type I Collagen Production in Fibrosis

Branko Stefanovic †, Heather A Michaels ‡, Adel Nefzi §,*
PMCID: PMC7958148  PMID: 33738075

Abstract

graphic file with name ml1c00006_0009.jpg

Fibrosis is a major medical problem caused by excessive synthesis of the extracellular matrix, composed predominantly of type I collagen, in various tissues. There are no approved antifibrotic drugs, and the major obstacle in finding clinically relevant compounds is the lack of specificity of current experimental drugs for type I collagen. Here we describe the discovery of a lead compound that specifically inhibited secretion of type I collagen by fibroblasts in culture at IC50 = 4.5 μM. The inhibition was specific for type I collagen, because secretion of fibronectin was not affected. In vitro, the compound inhibited binding of LARP6, the master regulator of translation of type I collagen mRNAs, to the 5′ stem-loop sequence element which regulates their translation. Because binding of LARP6 to collagen mRNAs is crucial for the development of fibrosis, this inhibitor represents a promising lead for optimization into specific antifibrotic drugs.

Keywords: Fibroproliferative disorders, Small molecule inhibitors, Fibrosis, Type I collagen, Combinatorial chemistry, Mixture-based libraries, Drug discovery, Solid-phase synthesis

Excessive production of extracellular matrix proteins is the hallmark of fibroproliferative diseases. The matrix is produced by activated fibroblasts and myofibroblasts in the affected organs. The most potent cytokine which activates these cells is TGFβ.1 Type I collagen is the most abundant protein of the fibrotic extracellular matrix and can account for up to 50% of the total proteins in a severely affected organ. The organs affected by fibrosis are liver, lungs, heart, kidneys, arterial walls, joints, skin, and intestine.2 The excessive collagen biosynthesis in fibrosis cannot be cured, and the process is progressive and debilitating. Fibroproliferative disorders are common, and on autopsy in the United States in 45% of cases some fibroproliferative process had been found.3

The approaches to discover treatments for fibrosis have been limited to four strategies: (1) removal of the profibrotic stimulus, (2) suppression of chronic inflammation which accompanies fibrosis, (3) reduction of the number and activation of collagen producing cells, and (4) stimulation of degradation of type I collagen.4−10 Although there have been some promising results in the short term animal models of fibrosis, specific antifibrotic drugs suitable for clinical use do not exist. It is surprising that the attempts to directly inhibit type I collagen biosynthesis have been lacking. Major complications of fibrosis can be attributable to excessive collagen deposition regardless of the nature of the profibrotic stimulus, type of collagen producing cells, or underlying inflammation.11 Therefore, an approach is needed that would discover compounds that can specifically inhibit excessive collagen synthesis, without significantly affecting the constitutive biosynthesis, because fibrosis is a chronic disease requiring long-term therapy. Small molecules with the ability to effectively and specifically inhibit excessive synthesis of type I collagen will represent first in class antifibrotic drugs which would improve the survival and quality of life of patients and significantly lessen the economic burden associated with this chronic disease affecting billions of people.

The research on regulation of type I collagen expression revealed that dramatically increased rate of type I collagen synthesis in fibrosis cannot be simply regarded as an augmentation of the constitutive synthesis, but that activation of additional mechanisms must take place.12−19 Type I collagen is a heterotrimeric protein assembled from the two α1(I) and one α2(I) polypeptides when they fold into a triple helix. After secretion of procollagen into the extracellular space, the terminal domains are removed by proteolytic cleavage and the rodlike triple helices of the central domain polymerize into fibrils and are covalently cross-linked.20 Type I collagen has a long half-life in normal tissues and is one of the most stable proteins. The half-life was measured to be between 4 and 12 months. The fractional synthesis rate of type I collagen (FSR, defined as percent of synthesis per day) is about 2% in the skin,21 while in the liver it is only 0.2%.22 This is unusually slow FSR and is significantly lower than the FSR of other proteins (18–140% per day).23 This is suggestive that the constitutive or replacement collagen synthesis is of very low rate.

In fibrosis, however, the synthesis of type I collagen, particularly in the areas affected by rapid fibrilogenesis, is dramatically increased.24,25 The regulatory step which is critical for the accelerated biosynthesis of type I collagen in fibrosis is binding of RNA binding protein LARP6 to the unique sequence element present in type I collagen mRNAs, the 5′ stem-loop (5′SL).13−17,26−30 The 5′SL is not found in any other mRNA, and the binding of LARP6 is needed to recruit accessory translational factors; when tethered, these proteins increase translational competency of collagen mRNAs and coordinate translation of α1(I) polypeptide to that of α2(I) polypeptide.13,14 The coordinated translation of collagen mRNAs results in production of both polypeptides in close proximity on the endoplasmic reticulum (ER) membrane, which increases their local concentration and accelerates their folding into type I collagen and rapid excretion into the extracellular matrix. The creation of 5′SL knock in mice revealed the clear distinction between profibrotic and constitutive regulation of type I collagen synthesis.31 A mutation was created in the collagen α1(I) gene of these animals, which altered the nucleotides encoding the 5′SL. The mutation did not change the coding region of the gene and the mRNA was expressed at normal levels. Thus, the homozygous 5′SL knock in mice encoded collagen α1(I) polypeptide without LARP6 dependent regulation from the mRNA that lacked 5′SL. The 5′SL knock in mice developed normally and have no abnormalities, demonstrating that constitutive collagen synthesis had not been affected. However, when liver fibrosis was induced in these animals, the degree of fibrosis was greatly reduced and their hepatic stellate cells, liver cells activated in fibrosis to produce large amounts of type I collagen, secreted only tracing amounts of type I collagen.31 These findings lead to hypothesis that it may be possible to selectively inhibit profibrotic regulation of type I collagen synthesis by disrupting the LARP6 dependent regulation.

In support of this premise, we have previously reported the discovery of one chemical compound that suppressed hepatic fibrosis in experimental animals by interfering with the LARP6 mechanism.32 This paper describes the synthesis, evaluation, and mechanism of action of novel compounds that can inhibit type I collagen production.

We screened the Torrey Pines (TP) collection of small molecule libraries which consists of more than 10 million compounds designed around 75 molecular scaffolds. Each library is arranged in positional scanning formats33,34 and contains an equal molar concentration of every compound of a given scaffold.35 In this manner, the different core scaffolds available in the TP collection were compared for their ability to reduce secretion of type I collagen from cultured cells. We added each library of the 75 scaffolds to human lung fibroblasts in culture at a concentration of 90 μg/mL. This high concentration was used because we were working with mixture-based libraries. Secretion of collagen α1(I) and α2(I) polypeptides into the cellular medium was measured by Western blot after 72 h. The prolonged incubation was done to select for the scaffold libraries which are not toxic to the cells because in preliminary trials some libraries showed substantial cell death after 72 h, and they were excluded from the analysis.

For analysis of collagen secretion, the cellular medium was replaced after 72 h with fresh medium and de novo collagen accumulation was allowed to proceed for 3 h. An aliquot of the medium was then directly analyzed by Western blot, while secretion of fibronectin was measured as the loading control. The libraries designated B8 and H3 have specifically reduced excretion of collagen α1(I) and α2(I) polypeptides, while the E5 and H3 libraries were less active and the D2, F5, and G2 libraries were inactive. Secretion of fibronectin was not affected by treatment of cells with these libraries, suggesting that they contain activity directed specifically forward type I collagen (Figure 1).

Figure 1.

Figure 1

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Pools of scaffolds screened for inhibition of type I collagen secretion. Left panel: the pools indicated at the bottom were added to a culture of human lung fibroblasts at 90 μg/mL, and secretion of collagen α1 (COL1A1) and α2 (COL1A2) polypeptides into cellular medium was measured by Western blot. FIB, fibronectin as loading control. Right panel: analysis repeated with lower concentrations of active pools.

Positional Scanning Screening

Based on these results, the library B8 corresponding to TP-2435 was selected for positional scanning screening. As described in Figure 2, the library TPI-2435 consists of two separate sublibraries having a single defined position (R) and one mixture position (X).36,37 The screening of the two sets of sublibraries each having 101 mixtures provided information about the most active groups of each variable position in the TPI-2435 library containing 10 200 (101 × 101) compounds. Each mixture was added at 10 μg/mL to human lung fibroblasts, and the secretion of collagen α1(I) polypeptide was measured in the cellular medium by Western blot. A portion of this screen is shown in Figure 3. Several mixtures were active in inhibiting type I collagen at 10 μg/mL. Three mixtures (F10, H4, and F3) in which the first position R1 was defined and six mixtures in which the R2 position was defined showed promising inhibition activity in the initial screen (indicated in red in Figure 3a).

Figure 2.

Figure 2

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Positional scanning deconvolution of the selected active library TPI-2435.

Figure 3.

Figure 3

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(a) Screening results of the positional scanning library TPI-2435. Identification of active mixtures. (b) Retesting of positive libraries from Figure 3a. Effect of expression of collagen α1(I) (COL1A1) and α2(I) (COL1A2) polypeptides and on fibronectin (FIB), as loading control.

For verification of this result, some mixtures that showed activity in the first screen were rescreened in a range of concentrations, together with fibronectin as a control for specificity (Figure 3b). Figure 4 shows the result with the mixture 2435-70 where the R1 position was defined as myristic acid and mixture 2435-167 with the R2 position defined as 4-phenylbutyric acid. The 2435-70 mixture showed strong inhibitory activity at 5 μg/mL; therefore, this scaffold with myristic acid as R1 was selected for the R2 positional scanning.

Figure 4.

Figure 4

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Inhibition of type I collagen secretion by mixture 2435-70 (R1: myristic acid) and mixture 2435-167 (R2: 4-phenylbutyric acid).

The 18 individual compounds named TPI-2659 derived from the PSL deconvolution of the TPI-2435 were prepared following the strategy described in Scheme 1.

Scheme 1. Parallel Solid Phase Synthesis of Individual Compounds TPI-2659 (the Same Strategy Was Used for the Synthesis of the Mixture-Based Library TPI-2435).

Scheme 1

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Preprepared Boc-l-Pro(4-N3)-OH (2S,4S) was coupled to the solid support, and the azide group was reduced in the presence of tin chloride in anhydrous dimethylformamide (DMF). The resulting amine was treated with different carboxylic acids in the presence diisopropylcarbodiimide (DIC) in anhydrous DMF. Following Boc removal, the amine was left to react with 4,6-dichloropyrimidine in dioxane at high temperature. The second chloro group was displaced by reaction with Boc-piperazine at high temperature. Following Boc removal, the free amine of the piperazine ring was acylated with various commercially available carboxylic acids in the presence of DIC and the final desired compounds were cleaved from the solid support, and then extracted, lyophilized and purified by preparative HPLC. The products were obtained in good yield and high purity and their identities were confirmed by LC-MS and NMR.

The inhibition of type I collagen production was tested in human lung fibroblasts. The cells were seeded at ∼80% confluency, and the compounds were added to the cellular medium at concentrations of 1–5 μM and incubated with cells for 18 h. The medium was changed, and fresh medium was added to the cells for 3 h. After 3 h of accumulation of type I collagen into this medium, the two polypeptides comprising type I collagen were measured by Western blot, as a readout of the potential of cells to produce type I collagen. The secretion of collagen polypeptides was normalized to secretion of fibronectin, as control for specificity.

The screening of TPI-2659 compounds at 5 μM showed significant differentiation in activity levels among the samples tested at each position, a key feature for deconvoluting a positional scanning library. As shown in Figure 5, nine compounds of TPI-2659 decreased collagen α1(I) polypeptide by >1 standard deviation of controls, while seven were inactive. The compound 2569-17 was the most active in this initial screen: it reduced collagen α1(I) polypeptide by >2 standard deviations of controls; therefore, it was selected for further evaluation.

Figure 5.

Figure 5

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Screening results of the individual compounds TPI-2659: The graph shows quantification of collagen α1(I) polypeptide after normalization to fibronectin. The standard deviation for control is indicated (n = 4).

Compound 2659-17 was further tested in a range of concentrations and by analyzing of both collagen polypeptides. This analysis showed that it inhibited secretion of collagen α2(I) polypeptide at 4 μM and α1(I) polypeptide at 5 μM (Figure 6). Thus, this compound represents a promising lead toward the development of specific drugs targeting excessive production of type I collagen and its mechanism of action was investigated.

To estimate IC50, two independent experiments were performed where the amount of collagen α1(I) and α2(I) polypeptides was measured by Western blot in cellular medium of cells treated with increasing concentrations of the compound 17. Fibronectin was also measured as loading control. The intensity of collagen signals was normalized to the intensity of fibronectin signals and plotted as arbitrary units (AU) against the #17 concentrations. From the linear fitting of data points the IC50 was estimated and an excellent agreement between the experiments was obtained. The IC50 for collagen α1(I) polypeptide (COL1A1) was 4.4 μM and 4.5 μM and for collagen α2(I) polypeptide (COL1A2) was 3.1 μM (Figure 6b).

Figure 6.

Figure 6

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(a) Activity of 2659-17 compound. (b) (A) IC50 for collagen α1(I) polypeptide. Collagen α1(I) polypeptide (COL1A1) was measured in cellular medium of cells treated with compound #17 in two independent experiments and normalized to the amount of fibronectin (COL1A1/FIB) and plotted as arbitrary units (AU) against #17 concentration. IC50 was estimated from the linear regression curves. (B) IC50 for collagen α2(I) polypeptide (COL1A2).

Mechanism of Action of 2659-17

LARP6 is one of the regulators of type I collagen production in fibrosis;28,32 therefore, we wanted to assess if compound 2659-17 inhibits type I collagen production by interfering with the LARP6 function. LARP6 functions by binding the unique 5′ stem-loop structure in type I collagen mRNAs (5′SL),27 so we tested if 2659-17 can inhibit the LARP6/5′SL binding. Two domains of LARP6, the La-domain and the RRM, together called the La-module, contribute to the high affinity of binding;30,38 however, the La-domain alone can also bind 5′SL, albeit with lower affinity. To assess if 2659-17 can inhibit the binding of La-module and La-domain, we prepared these recombinant proteins, bound them to fluorescently labeled 5′SL RNA, and added 2659-17 to these in vitro binding reactions. The formation of protein/RNA complexes was monitored by gel mobility shift assay (Figure 7). The 2659-17 compound inhibited binding of the recombinant La-domain to 5′SL RNA at a concentration of 1 μM or higher (Figure 7, left panel) and that of the La-module at a concentration of 2.5 μM or higher. The higher concentration needed for the La-module inhibition probably reflects the higher affinity by which this recombinant protein binds 5′SL RNA. The gel in Figure 7 clearly indicates that LARP6 forms a complex with its target RNA, the 5′ stem-loop sequence present in type I collagen mRNAs. In the left panel, there is a band of unbound RNA and a band of La domain of LARP6 bound to the RNA. By adding increasing concentrations of the 2659-17 compounds, the intensity of La-domain bound band decreases, indicating the dissociation of the protein from the RNA and the intensity of the free RNA band increases. In the right panel, the binding of La-module of LARP6, which contains the La-domain and the RRM domain together and which binds the 5′ stem-loop RNA with higher affinity, is shown. The La-module tends to form dimers with itself and two bands of La-module bound to the RNA are visible, the La-module monomer/RNA and La-module dimer/RNA. With increasing concentrations of 2659-17, both complexes dissociate. This clearly indicates that 2659-17 can dissociate LARP6 from its target RNA. LARP6 has no other target RNA known to test if such an interaction will also be dissociated.

Figure 7.

Figure 7

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Inhibition of LARP6 binding to 5′SL of collagen mRNAs. Gel mobility shift assay. Left panel: inhibition of binding of La-domain of LARP6 to collagen 5′ stem-loop RNA. RNA/LA COMPLEX: La domain bound to RNA. RNA: unbound RNA. Right panel: inhibition of binding of La-module of LARP6 to collagen 5′ stem-loop RNA. RNA/LA MODULE COMPLEX 1: monomer of La-module bound to RNA, RNA/LA MODULE COMPLEX 2, dimer of La-module bound to RNA. RNA: unbound RNA.

From these experiments, we concluded that the likely mechanism of action of 2659-17 is inhibition of the interaction of LARP6 with collagen mRNAs. Because this interaction is unique for type I collagen mRNAs and critical for high type I collagen production in fibrosis, 2659-17 may represent the new lead compound for development of specific antifibrotic drugs.

Excessive synthesis of type I collagen is the main pathological process in fibrosis. However, there are no small molecules that would directly inhibit type I collagen biosynthesis.39 Here we describe discovery of a compound that can suppress type I collagen production by lung fibroblasts and which acts by inhibiting the binding of LARP6 to the unique regulatory element of collagen mRNAs, the 5′ stem-loop (5′SL).12 This compound was discovered by positional screening and deconvolution of small set of chemical scaffolds. The screening of relatively small number of scaffolds in each round of screening allowed us to directly measure type I collagen production by Western blot to assess the effect. Such phenotypic screen was specific, we measured both collagen polypeptides which comprise type I collagen for cross-validation of the results and analyzed fibronectin, another extracellular matrix protein, to verify the specificity for type I collagen.

The 2659-17 compound, which was the most active, inhibited type I collagen production by lung fibroblasts at concentrations of 4–5 μM, making it a promising therapeutic lead for development of more potent compounds. It can clearly penetrate the cells in culture, and we have not observed any adverse effects on cell viability at the effective concentrations. In vitro, it inhibited the binding of LARP6 to the 5′SL, suggesting that this may be its mechanism of action. LARP6 binds 5′SL of type I collagen mRNAs with high affinity and strict sequence specificity.27,30 Two domains of LARP6 are involved in 5′SL binding; the La-domain recognizes the sequence and interacts with 5′SL RNA with the low affinity, while the RRM domain increases the stability of the LARP6/5′SL complex. Together, these domains are termed the La-module.40 The 2659-17 compound showed inhibition of in vitro binding of the La-domain at 1 μM and that of La-module at 2.5 μM, which correlates with the difference in their binding affinities. These concentrations are close to the concentrations needed in the cellular experiment. The fact that 2659-17 inhibited binding of the La-domain alone suggests that it has a binding site in this domain. For the full elucidation of the mechanism of action of 2659-17 and the ensuing derivatives, the binding pocket in La-domain has to be determined.

Another LARP6 inhibitor, C9, was recently described.32 C9 has a different structure for 2659-17, although it showed a similar mechanism of action. C9 was tested in animal models of hepatic fibrosis, and it suppressed fibrosis development when administered in doses of 1 mg/kg into experimental animals. This suggests that the derivatives of 2659-17 may have similar potency and that multiple LARP6 inhibitors can be discovered to broaden the spectrum of candidate antifibrotic drugs. In summary, this work reports the discovery of a promising lead compound that suppresses type I collagen production by fibroblasts in culture in low micromolar concentrations and acts as LARP6 binding inhibitor. The approach described here can be used for further optimization of 2659-17 before advancing the compounds into preclinical studies.

Acknowledgments

Florida Department of Health.

Glossary

Abbreviations Used

TGFβ

transforming growth factor beta

FSR

fractional synthesis rate

RNA

ribonucleic acid

LARP6

La ribonucleoprotein 6

mRNA

messenger RNA

Boc

tertbutyloxycarbonyl

HPLC

high-performance liquid chromatography

LC-MS

liquid chromatography–mass spectrometry

NMR

nuclear magnetic resonance.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00006.

  • 1H NMR data of active compounds 2659-17; LCMS of all 18 individual compounds TPI-2659; description of the synthetic strategy of individual compounds TPI-2659 and library TPI-2435 library distribution (PDF)

The authors declare no competing financial interest.

Supplementary Material

ml1c00006_si_001.pdf (1.2MB, pdf)

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