Computational Investigation of a Series of Small Molecules as Potential Compounds for Lysyl Hydroxylase-2 (LH2) Inhibition.

The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O2 tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O2-transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.

Collagen cross-linking mediated by lysyl hydroxylase 2: an enzymatic battlefield to combat fibrosis.

The hallmark of fibrosis is an excessive accumulation of collagen, ultimately leading to organ failure. It has become evident that the deposited collagen also exhibits qualitative modifications. A marked modification is the increased cross-linking, leading to a stabilization of the collagen network and limiting fibrosis reversibility. Not only the level of cross-linking is increased, but also the composition of cross-linking is altered: an increase is seen in hydroxyallysine-derived cross-links at the expense of allysine cross-links. This results in irreversible fibrosis, as collagen cross-linked by hydroxyallysine is more difficult to degrade. Hydroxyallysine is derived from a hydroxylysine in the telopeptides of collagen. The expression of lysyl hydroxylase (LH) 2 (LH2), the enzyme responsible for the formation of telopeptidyl hydroxylysine, is universally up-regulated in fibrosis. It is expected that inhibition of this enzyme will lead to reversible fibrosis without interfering with the normal repair process. In this review, we discuss the molecular basis of collagen modifications and cross-linking, with an emphasis on LH2-mediated hydroxyallysine cross-links, and their implications for the pathogenesis and treatment of fibrosis.

Development of a High-Throughput Lysyl Hydroxylase (LH) Assay and Identification of Small-Molecule Inhibitors against LH2.

Lysyl hydroxylase-2 (LH2) catalyzes the hydroxylation of telopeptidyl lysine residues on collagen, leading to the formation of stable collagen cross-links that connect collagen molecules and stabilize the extracellular matrix. High levels of LH2 have been reported in the formation and stabilization of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), leading to fibrosis and cancer metastasis in certain tissues. Identification of small-molecule inhibitors targeting LH2 activity requires a robust and suitable assay system, which is currently lacking. Thus, despite being a promising target for these diseases, small-molecule inhibitors for LH2 have yet to be reported. Therefore, we developed a luminescence-based strategy to monitor LH activity and validated its ability to identify new inhibitors in a screen of approximately 65,000 compounds against LH2. Primary hits were confirmed using the same LH assay against mimiviral L230. This newly developed LH assay is robust, suitable for high-throughput screening, and able to identify potent specific inhibitors of LH2.

Lysyl Hydroxylase Inhibition by Minoxidil Blocks Collagen Deposition and Prevents Pulmonary Fibrosis via TGF-ß1/Smad3 Signaling Pathway.

BACKGROUND Idiopathic pulmonary fibrosis (IPF) is a deadly disease characterized by excessive collagen in the extracellular matrix (ECM) of the lungs. Collagen is the primary protein component of the ECM. However, the exact mechanisms underlying the formation and deposition of collagen in the ECM under normal and pathological conditions remain unclear. Previous studies showed that lysyl hydroxylase (LH) plays a crucial role in the formation of collagen. Minoxidil is an FDA-approved anti-hypertensive agent that inhibits LH that reduces fibrosis. In this study, we investigated the functional roles of LHs (LH1, LH2, and LH3) in pulmonary fibrosis and the anti-fibrotic effects of minoxidil. MATERIAL AND METHODS Patient serum samples were examined for their expression of procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLOD) 1-3, the genes encoding LH 1-3. Mice with bleomycin (BLM 2.5 mg/kg)-induced pulmonary fibrosis were administered a minoxidil solution (30 mg/kg) by oral gavage. RESULTS The PLOD mRNA levels were significantly higher in the IPF patients than in the healthy control subjects. Minoxidil suppressed the BLM-induced pulmonary fibrosis in vivo. These effects were associated with blocking TGF-ß1/Smad3 signal transduction and attenuating the expression and activity of LHs, resulting in decreased collagen formation, thus reducing the pulmonary fibrosis. The anti-fibrotic effects of minoxidil may be mediated through competitive inhibition of LHs activity, resulting in decreased pyridine cross-link formation and collagen production and deposition. CONCLUSIONS The results of this study suggest that LH represents a target to prevent or treat pulmonary fibrosis, and minoxidil may provide an effective agent to inhibit LHs.

Nanoscale dysregulation of collagen structure-function disrupts mechano-homeostasis and mediates pulmonary fibrosis.

Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.

KDM1A regulated the osteo/dentinogenic differentiation process of the stem cells of the apical papilla via binding with PLOD2.

OBJECTIVES: Dental tissue-derived mesenchymal stem cells (MSCs)-mediated pulp-dentin regeneration is considered a potential approach for the regeneration of damaged teeth. Enhancing MSC-mediated pulp-dentin regeneration is based on an understanding of the molecular mechanisms underlying directed cell differentiation process. Histone demethylation enzyme, lysine demethylase 1A (KDM1A) can regulate the differentiation of some MSCs, but its role in dental tissue-derived MSCs is unclear. MATERIAL AND METHODS: We obtained SCAPs from immature teeth. Alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative calcium analysis, osteogenesis-related genes expression and in vivo transplantation experiment were used to explore the osteo/dentinogenic differentiation. Co-immunoprecipitation (Co-IP) assay was used to investigate the binding protein. RESULTS: Knock-down of KDM1A reduced ALP activity and mineralization, promoted the expressions of osteo/dentinogenic differentiation markers DSPP, DMP1, BSP and key transcript factors, RUNX2, OSX, DLX2 in SCAPs, and also enhanced the osteo/dentinogenesis in vivo. In addition, KDM1A could associate with PLOD2 to form protein complex. And knock-down of PLOD2 inhibited ALP activity and mineralization, and promoted the expressions of DSPP, DMP1, BSP, RUNX2, OSX and DLX2 in SCAPs. CONCLUSIONS: KDM1A might have different role in different stages of osteo/dentinogenic differentiation process by binding partner with PLOD2, and finally resulted in the inhibited function for the osteo/dentinogenesis in SCAPs. Our studies provided a further understanding of the regulatory mechanisms of dynamic osteo/dentinogenic differentiation process in dental tissue MSCs.

A strong developmental isotope effect in Caenorhabditis elegans induced by 5,5-deuterated lysine.

Effects exerted by heavy isotope substitution in biopolymers on the functioning of whole organisms have not been investigated. We report on the decrease of permissive temperature of nematodes fed with bacteria containing 5,5-D2-lysine. We synthesized 5,5-dideuterolysine and, taking advantage of lysine being an essential amino acid, showed that C. elegans with modified lysine poorly develop from larvae into fertile adult hermaphrodites. This effect occurs only at high temperature within the permissible range for C. elegans (25 °C) and completely vanishes at 15 °C. The only known metabolic involvement of C5 in lysine is in post-translational modification through lysyl hydoxylases. Indeed, siRNA experiments showed that deficiency of let-268/plod lysyl-hydroxylase/glycosydase further amplifies the isotope effect making it apparent even at 20 °C, whereas control siRNAs as well as another lysyl-hydroxylase (psr-1/jmjdD) siRNA do not. We report for the first time that a site-specific deuteration may strongly affect the development of the whole animal organism especially under the conditions of deficiency of the corresponding enzyme. These findings provide the basis for our ongoing efforts to employ isotope effects for fine tuning of metabolic pathways to mitigate pathological processes.

Essential roles of insulin, AMPK signaling and lysyl and prolyl hydroxylases in the biosynthesis and multimerization of adiponectin.

Post-translational modifications (PTMs) of the adiponectin molecule are essential for its full bioactivity, and defects in PTMs leading to its defective production and multimerization have been linked to the mechanisms of insulin resistance, obesity, and type-2 diabetes. Here we observed that, in differentiated 3T3-L1 adipocytes, decreased insulin signaling caused by blocking of insulin receptors (InsR) with an anti-InsR blocking antibody, increased rates of adiponectin secretion, whereas concomitant elevations in insulin levels counteracted this effect. Adenosine monophosphate-activated protein kinase (AMPK) signaling regulated adiponectin production by modulating the expression of adiponectin receptors, the secretion of adiponectin, and eventually the expression of adiponectin itself. We found that lysyl hydroxylases (LHs) and prolyl hydroxylases (PHs) were expressed in white-adipose tissue of ob/ob mice, wherein LH3 levels were increased compared with controls. In differentiated 3T3-L1 adipocytes, both non-specific inhibition of LHs and PHs by dipyridyl, and specific inhibition of LHs by minoxidil and of P4H with ethyl-3,4-dihydroxybenzoate, caused significant suppression of adiponectin production, more particularly of the higher-order isoforms. Transient gene knock-down of LH3 (Plod3) caused a suppressive effect, especially on the high molecular-weight (HMW) isoforms. These data indicate that PHs and LHs are both required for physiological adiponectin production and in particular are essential for the formation/secretion of the HMW isoforms.

Hypoxia signaling--license to metastasize.

Hypoxia-inducible factors (HIF) have long been linked to malignant tumor phenotypes in various cancer types, and several downstream mediators of HIF action are deregulated in metastatic carcinomas. A new study links hypoxia-induced collagen remodeling to sarcoma progression, providing evidence for unifying mechanisms of carcinoma and sarcoma metastasis.

Hypoxia-dependent modification of collagen networks promotes sarcoma metastasis.

UNLABELLED: Intratumoral hypoxia and expression of hypoxia-inducible factor-1α (HIF-1α) correlate with metastasis and poor survival in patients with sarcoma. We show here that hypoxia controls sarcoma metastasis through a novel mechanism wherein HIF-1α enhances expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). We show that loss of HIF-1α or PLOD2 expression disrupts collagen modification, cell migration, and pulmonary metastasis (but not primary tumor growth) in allograft and autochthonous LSL-Kras(G12D/+); Trp53(fl/fl) murine sarcoma models. Furthermore, ectopic PLOD2 expression restores migration and metastatic potential in HIF-1α-deficient tumors, and analysis of human sarcomas reveals elevated HIF1A and PLOD2 expression in metastatic primary lesions. Pharmacologic inhibition of PLOD enzymatic activity suppresses metastases. Collectively, these data indicate that HIF-1α controls sarcoma metastasis through PLOD2-dependent collagen modification and organization in primary tumors. We conclude that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell dissemination. SIGNIFICANCE: Undifferentiated pleomorphic sarcoma (UPS) is a commonly diagnosed and particularly aggressive sarcoma subtype in adults, which frequently and fatally metastasizes to the lung. Here, we show the potential use of a novel therapeutic target for the treatment of metastatic UPS, specifi cally the collagen-modifying enzyme PLOD2.

Mechanism of inhibition of lysyl hydroxylase activity by the organophosphates malathion and malaoxon.

Direct inhibition of lysyl hydroxylase by malathion and malaoxon was observed in an in vitro enzyme assay with recombinant lysyl hydroxylase expressed via a baculoviral system. The IC50 values for malathion and malaoxon were estimated to be approximately 60 and 45 mM, respectively. Additional kinetic studies showed this inhibition to be competitive or partially competitive with respect to the synthetic (collagen) peptide, partially uncompetitive with respect to Fe(2+), and partially noncompetitive with respect to ascorbic acid. The calculated values for the K(i) were consistent with the IC50 values. Allosteric effects were not found for any of the cofactors tested, the peptide substrate, or the inhibitors. Interactions were found to be unimolecular for lysyl hydroxylase and its substrate and cofactors as well as for the inhibitors malathion and malaoxon. A computer search of a protein structure database showed an unexpected region of partial homology between the active site sequence of acetylcholinesterase and a segment of lysyl hydroxylase, suggesting a possible molecular basis for these observations. These results suggest the possibility of a novel and hitherto unexpected class of inhibitors of lysyl hydroxylase, based on the organophosphate structure, that might be of value for testing as antifibrotic drugs.

Inhibition of lysyl hydroxylase by malathion and malaoxon.

The inhibition of lysyl hydroxylation in newly synthesized collagen by malathion and its oxidation product malaoxon were studied with cultured rat fetal lung fibroblasts. Exposure of these cells to 125 microM malathion or malaoxon for 96 h resulted in a 25 and a 30% decrease in the ratio of hydroxylated lysine/lysine residues, respectively, in acid hydrolyzed cell lysates compared to control values. This relative decrease in hydroxylysine was not caused by cytotoxicity or changes in total collagen content, which were found to remain constant as measured by Alamar Blue metabolism and Sircol dye binding assays. Direct inhibition of lysyl hydroxylase by malathion and malaoxon was observed using an in vitro enzyme assay with recombinant lysyl hydroxylase with a baculoviral system. The IC50 values for malathion and malaoxon were estimated to be approximately 60 and 45 microM, respectively. These observed IC50 values are consistent with calculated values for the intracellular concentration of malathion in the cell culture experiments. These results support a significant role for inhibition of lysyl hydroxylase activity as causing, or contributing to, the teratogenic effects of malathion and malaoxon.

Effect of minoxidil on rabbit lens epithelial cell behavior in vitro and in situ.

BACKGROUND: Lens epithelial cell (LEC) proliferation and the associated production of extracellular matrix (ECM) are responsible for capsular opacification after cataract-IOL surgery. Minoxidil is an inhibitor of lysyl hydroxylase, an enzyme involved in procollagen hydroxylation. To evaluate the potential efficacy of minoxidil in inhibiting postoperative capsular opacification, we examined the effects of minoxidil on LEC behavior in cell and organ cultures. METHODS: We examined minoxidil effects on collagen production, migration and proliferation of cultured rabbit LECs as well as its ultrastructural effects, and also its effects on the cell population in organ-cultured capsular bag. RESULTS: No cytotoxicity was identified by MTT assay at the concentrations up to 3.0 mM of minoxidil, whereas it decreased the collagen production in LECs. Minoxidil also inhibited migration and proliferation of cells. Ultrastructural observation revealed the presence of dilated endoplasmic reticulum in LECs treated with minoxidil, indicating the accumulation of protein, probably underhydroxylated collagen precursors. The capsules cultured with minoxidil appeared less opaque than control specimens. On histological examination the numbers of cells on equatorial capsules were found to be significantly lower in minoxidil culture than in control culture. No lens cells were detected on the central posterior capsule of minoxidil culture, whereas they were seen in control. CONCLUSION: Minoxidil inhibited LEC migration and proliferation in vitro, as well as collagen secretion. Collagen secretion may be essential for LEC migration and proliferation. Minoxidil also attenuated repopulation of LECs on the inner surface of organ-cultured capsules. Minoxidil may be a potential inhibitor of postoperative capsular opacification.

Lysyl hydroxylase 3 is a multifunctional protein possessing collagen glucosyltransferase activity.

Lysyl hydroxylase (EC ) and glucosyltransferase (EC ) are enzymes involved in post-translational modifications during collagen biosynthesis. We reveal in this paper that the protein produced by the cDNA for human lysyl hydroxylase isoform 3 (LH3) has both lysyl hydroxylase and glucosyltransferase (GGT) activities. The other known lysyl hydroxylase isoforms, LH1, LH2a, and LH2b, have no GGT activity. Furthermore, antibodies recognizing the amino acid sequence of human LH3 and those against a highly purified chicken GGT partially inhibited the GGT activity. Similarly, a partial inhibition was observed when these antibodies were tested against GGT extracted from human skin fibroblasts. In vitro mutagenesis experiments demonstrate that the amino acids involved in the GGT active site differ from those required for LH3 activity.

Minoxidil-induced alteration of corneal topography after radial keratotomy.

PURPOSE: To determine the antiproliferative effect of minoxidil on human corneal epithelium (hCE) proliferation in vitro and to assess whether topical minoxidil can significantly alter corneal topography after radial keratotomy (RK) by inhibiting myofibroblast activity in the keratotomy wound. SETTING: Corneal Research Laboratory, University of Chicago, Illinois, USA. METHODS: In the in vitro evaluation, proliferating hCE was exposed to minoxidil (0.1 to 2.0 mM) for 96 hours to determine the minimum inhibitory dose. Human corneal epithelium cell proliferation was assessed by the incorporation of bromodeoxyuridine (BRDU) into DNA. In the in vivo analysis, eight New Zealand albino rabbits had an eight-incision bidirectional RK on one eye and were divided into two groups. The control eyes (n = 3) received tobramycin and dexamethasone (TobraDex), ciprofloxacin hydrochloride (Ciloxan), and balanced salt solution (BSS) drops four times a day for 3 weeks, while the treatment eyes (n = 5) received TobraDex, Ciloxan, and minoxidil 1.0 mM drops four times daily for 3 weeks. The net change in corneal curvature at 3 weeks was analyzed with corneal topography. Myofibroblast activity in the keratotomy wound was assessed using alpha smooth muscle actin staining techniques. RESULTS: At concentrations of 1.0 mM and above, minoxidil caused a statistically significant, dose-dependent reduction in hCE cellular proliferation ranging from 29 to 44% (P < .05). Minoxidil (1.0 mM) caused a statistically significant central corneal flattening effect of 4.66 diopters (D) after RK in the treatment eyes compared with 1.11 D in the control eyes (P = .05). Histologically, minoxidil-treated keratotomy wounds lacked cells with contractile elements consistent with myofibroblast differentiation. Corneal epithelial wound healing was similar in both groups. CONCLUSION: At the appropriate dose, topical minoxidil may be a useful adjunctive treatment that can reduce the number of undercorrections after mini-RK without apparent toxicity to the corneal epithelium.

A minoxidil-related compound lacking a C6 substitution still exhibits strong anti-lysyl hydroxylase activity in vitro.

It has been previously reported that minoxidil inhibits the activity of lysyl hydroxylase (LH), an enzyme which catalyzes the formation of hydroxylysine, which is necessary for proper maturation of collagen at the transcriptional and enzymatic levels. Using the reverse transcriptase-polymerase chain reaction, we confirmed that this inhibition occurred at least at the transcriptional level. Furthermore, we took advantage of this sensitive and rapid method to perform a quantitative structure activity relation study using several compounds structurally related to minoxidil. We found that when the C6 of the pyrimidinyl moiety was substituted, it had to be by a tertiary nitrogen, i.e. an N-piperidin ring for the inhibition of LH mRNA synthesis to be observed. Surprisingly, however, we found that 2,4-diamino-pyrimidin-3-oxide, a new compound lacking an organic moiety para to the nitroxide oxygen, also retained a high inhibitory effect on LH mRNA expression, comparable to that of minoxidil. We thus conclude that the presence of a substituent para to the nitroxide oxygen is dispensable for inhibition of LH mRNA to be observed in vitro. This brings new insights into the design of therapeutic agents useful in any condition where an overproduction of mature collagen is unwanted, i.e. accelerated wound healing, keloids and localized scleroderma.

Effect of lysyl hydroxylase inhibitor, minoxidil, on ultrastructure and behavior of cultured rabbit subconjunctival fibroblasts.

BACKGROUND: Minoxidil is an inhibitor of lysyl hydroxylase, an enzyme involved in collagen production, and decreases collagen production in vitro. We investigated the in vitro effects of minoxidil on behavior such as proliferation and migration of rabbit subconjunctival fibroblasts (SCFs). The ultrastructural effect of the drug on SCFs was also examined. METHODS: Proliferation of SCFs and closure of the defect produced in monolayer cultures in the presence or absence of minoxidil was studied. The ultrastructure of SCFs treated with minoxidil was also examined. RESULTS: Minoxidil inhibited SCF proliferation and the closure of the defect produced in monolayer cell sheets. Ultrastructural observations revealed extensive areas of irregularly dilated endoplasmic reticulum in cells treated with minoxidil, indicating the accumulation of protein, probably underhydroxylated collagen precursors, in the cisternae of endoplasmic reticulum. CONCLUSIONS: The results indicated that minoxidil attenuated cellular activities of SCFs such as proliferation and migration in vitro. The exact mechanism of the inhibitory effects of minoxidil on these cellular activities is unknown. The findings suggest that the drug might help to prevent bleb scarring after glaucoma filtering surgery.

Prolyl hydroxylase inhibitor and lysyl hydroxylase inhibitor inhibit spreading of corneal epithelium.

BACKGROUND: The spreading of epithelium is critical in the healing of corneal wounds. Such epithelial spreading requires the continuous production of protein and glycoprotein. To determine whether collagen production is required for the spreading of corneal epithelium, we studied the effects of inhibitors for collagen production on spreading of corneal epithelium in vitro. METHODS: We examined the effect of two proline analogs, L-azetidine 2-carboxylic acid and cis-hydroxyproline, a prolyl hydroxylase inhibitor, ethyl-3,4-dihydroxybenzoate, and a lysyl hydroxylase inhibitor, minoxidil, on the spreading of epithelium of organ-cultured rabbit cornea. RESULTS: Both analogs and inhibitors inhibited epithelial spreading in a dose-dependent manner. CONCLUSION: These observations indicate that collagen production may be involved in the spreading of corneal epithelium.

Inhibition of cultured human RPE cell proliferation and lysyl hydroxylase activity by hydroxy derivatives of minoxidil.

PURPOSE: To examine the antiproliferative and lysyl hydroxylase suppressing effects of 3'-hydroxyminoxidil and 4'-hydroxyminoxidil, derivatives of minoxidil devoid of the antihypertensive effect, on human retinal pigment epithelial (RPE) cells in culture. METHODS: Subconfluent and confluent cultures of RPE cells, exposed to 0.01 to 5 mM 3' or 4'-hydroxyminoxidil for 15 minutes to 7 days, were examined for proliferation, viability, and morphologic changes. Lysyl hydroxylase activity in confluent cultures exposed to 1 mM 3'- or 4'-hydroxyminoxidil was determined by measuring the amount of 3H2O released from L-(4,5-3H)lysine-labeled unhydroxylated procollagen substrate after vacuum distillation. RESULTS: Both compounds inhibited the proliferation of subconfluent cultures of RPE cells in a dose-dependent fashion. The 50% effect occurred at 0.25 mM for 3'-hydroxyminoxidil and 0.5 mM for 4'-hydroxyminoxidil. The antiproliferative effect was detectable within 24 hours, required a minimum 1-hour exposure, and persisted even after the drug was removed from the culture medium. Cell viability experiments provided no evidence for toxicity. In contrast, the number of cells at confluent density was not affected. Both 3'-hydroxyminoxidil and 4'-hydroxyminoxidil suppressed lysyl hydroxylase activity by 72%. CONCLUSIONS: The structure of minoxidil can be altered to reduce the antihypertensive activity while preserving the antiproliferative and lysyl hydroxylase suppressing effects. The hydroxy derivatives of minoxidil may be useful in the treatment of proliferative vitreoretinopathy, a disease with unwanted proliferation of RPE cells.

Minimum structural requirements for minoxidil inhibition of lysyl hydroxylase in cultured fibroblasts.

The structural features of minoxidil contributing to its inhibitory effect on lysyl hydroxylase in cultured fibroblasts were investigated. Minoxidil analogs in which the pyrimidine ring (two nitrogens) was replaced with a pyridine ring (one nitrogen) or a sym-triazine ring (three nitrogens) suppressed lysyl hydroxylase activity without affecting prolyl hydroxylase activity, as did an analog in which the piperidine ring was replaced with an N,N-diethyl group. By contrast, minoxidil analogs bearing an N-monoalkyl (ethyl or butyl) group in place of the piperidine substituent failed to suppress lysyl hydroxylase activity. The results indicate that nitroxides of pyridine and triazine, in addition to pyrimidine, having an ortho amino group can act as specific inhibitors of lysyl hydroxylase in the cell. The minimum structural requirement for inhibitory activity appears to be an organic moiety containing a tertiary nitrogen para to the nitroxide oxygen, a condition that is best fulfilled by the piperidine ring in minoxidil. Hydroxylation of minoxidil at the 3 or 4 position of the piperidine ring had no impact on its ability to inhibit the post-translational hydroxylation of lysine during collagen biosynthesis. Fibroblasts treated with minoxidil, 3'-hydroxyminoxidil, or 4'-hydroxyminoxidil synthesized a collagen specifically deficient in hydroxylysine by approximately 70%, which completely accounted for the diminished lysyl hydroxylase activity. The percentage of total proteins synthesized as collagen was reduced minimally by minoxidil but not by 3'- or 4'-hydroxyminoxidil. The studies offer a potential means for therapeutic intervention of excessive collagen deposition during fibrosis, using minoxidil or preferably its hydroxy derivatives to limit the supply of hydroxylysine for collagen crosslink formation.