Expression of the Discoidin Domain Receptor Family Depended on Glucose and Their High Expression in Arterial Tissues in the Rat Model of Type 2 Diabetes.

The active form of discoidin domain receptors (DDRs) is expressed in cell surface and regulated post-translationally by glucose. The DDR2 and DDR1 transfected in HEK293 cells were expressed mainly in their active forms with sizes of 130 and 120 kDa, respectively. DDRs were observed predominantly as 100 kDa proteins in glucose-depleted culture conditions. However, transfection of endothelial growth factor receptor (EGFR) in HEK293 cells resulted in the expression of only one form regardless of glucose concentration. Vascular smooth muscle cells, HT1080s, and MDA-MB-231 cancer cells expressed DDRs in their active forms in high glucose concentrations, which did not occur with EGFR. In diabetic rats, DDRs were expressed at high levels in arterial tissue but EGFR was not highly expressed. Taken together, these results suggest that DDRs expression depends on glucose concentration it may cooperate in the development of atherosclerosis and kidney fibroblasts, promoting nephropathy in diabetic rats.

Identification of Leishmania donovani PEX5-PTS1 Interaction Inhibitors through Fluorescence Polarization-Based High-Throughput Screening.

Leishmaniasis, an infectious disease caused by pathogenic Leishmania parasites, affects millions of people in developing countries, and its re-emergence in developed countries, particularly in Europe, poses a growing public health concern. The limitations of current treatments and the absence of effective vaccines necessitate the development of novel therapeutics. In this study, we focused on identifying small molecule inhibitors which prevents the interaction between peroxin 5 (PEX5) and peroxisomal targeting signal 1 (PTS1), pivotal for kinetoplastid parasite survival. The Leishmania donovani PEX5, containing a C-terminal tetratricopeptide repeat (TPR) domain, was expressed and purified, followed by the quantification of kinetic parameters of PEX5-PTS1 interactions. A fluorescence polarization-based high-throughput screening assay was developed and small molecules inhibiting the LdPEX5-PTS1 interaction were discovered through the screening of a library of 51,406 compounds. Based on the confirmatory assay, nine compounds showed half maximal inhibitory concentration (IC50) values ranging from 3.89 to 24.50 µM. In silico docking using a homology model of LdPEX5 elucidated that the molecular interactions between LdPEX5 and the inhibitors share amino acids critical for PTS1 binding. Notably, compound P20 showed potent activity against the growth of L. donovani promastigotes, L. major promastigotes, and Trypanosoma brucei blood stream form, with IC50 values of 12.16, 19.21, and 3.06 µM, respectively. The findings underscore the potential of targeting LdPEX5-PTS1 interactions with small molecule inhibitors as a promising strategy for the discovery of new anti-parasitic compounds.

Repurposing of conformationally-restricted cyclopentane-based AKT-inhibitors leads to discovery of potential and more selective antileishmanial agents than miltefosine.

Conformational restriction was addressed towards the development of more selective and effective antileishmanial agents than currently used drugs for treatment of Leishmania donovani; the causative parasite of the fatal visceral leishmaniasis. Five types of cyclopentane-based conformationally restricted miltefosine analogs that were previously explored in literature as anticancer AKT-inhibitors were reprepared and repurposed as antileishmanial agents. Amongst, positions-1 and 2 cis-conformationally-restricted compound 1a and positions-2 and 3 trans-conformationally-restricted compound 3b were highly potent eliciting sub-micromolar IC50 values for inhibition of infection and inhibition of parasite number compared with the currently used miltefosine drug that showed low micromolar IC50 values for inhibition of infection and inhibition of parasite number. Compounds 1a and 3b eradicated the parasite without triggering host cells cytotoxicity over more than one log concentration interval which is a superior performance compared to miltefosine. In silico studies suggested that conformational restriction conserved the conformer capable of binding LdAKT-like kinase while it might be possible that it excludes other conformers mediating undesirable effects and/or toxicity of miltefosine. Together, this study presents compounds 1a and 3b as antileishmanial agents with superior performance over the currently used miltefosine drug.

Rational repurposing, synthesis, in vitro and in silico studies of chromone-peptidyl hybrids as potential agents against Leishmania donovani.

A chromone-peptidyl hybrids series was synthesised and rationally repurposed towards identification of potential antileishmanial hits against visceral leishmaniasis. Three hybrids 7c, 7n, and 7h showed potential IC50 values (9.8, 10, and 12 µM, respectively) which were comparable to erufosine IC50 (9.8 µM) but lower potency than miltefosine IC50 (3.5 µM). Preliminary assessment of cytotoxicity using human THP-1 cells presented chromone-peptidyl hybrids 7c and 7n as non-cytotoxic up to 100 µM while erufosine and miltefosine had CC50 of 19.4 µM and >40 µM, respectively. In silico studies pinpointed the N-p-methoxyphenethyl substituent at the peptidyl moiety together with the oxygen-based substituted functions of the phenyl ring of the chromone moiety as crucial players in binding to LdCALP. Together, these findings present chromone-peptidyl hybrids 7c and 7n as potential and anticipated non-cytotoxic antileishmanial hit compounds for possible development of potential antileishmanial agents against visceral leishmaniasis.

Discovery of novel Leishmania major trypanothione synthetase inhibitors by high-throughput screening.

Leishmaniasis is an infectious disease caused by obligate intracellular protozoa of the genus Leishmania with high infection and death rates in developing countries. New drugs with better pharmacological performance with regards to safety, efficacy, toxicity, and drug resistance than those/the ones currently used are urgently needed. Trypanothione synthetase (TryS) is an attractive target for the development of drugs against leishmaniasis because it is specific and essential to kinetoplastid parasites. In this study, Leishmaniamajor TryS was expressed and purified, and the kinetic parameters of purified TryS were determined. To identify novel inhibitors of LmTryS, a high-throughput screening (HTS) assay was developed and used to screen a library of 35,040 compounds. In the confirmatory assay, 42 compounds displayed half maximal inhibitory concentration (IC50) values < 50 µM and six of them corresponded to novel structures with IC50 ranging from 9 to 19 µM against LmTryS enzyme activity. Of the six inhibitors, TS001 showed the highest activity against growth of L. major promastigotes, L. donovani promastigotes, and Trypanosoma brucei brucei Lister 427 with IC50 values of 17, 26, and 31 µM, respectively. An in silico docking study using a homology model of LmTryS predicted the molecular interactions between LmTryS and the inhibitors.

Discovery of Leishmania donovani topoisomerase IB selective inhibitors by targeting protein-protein interactions between the large and small subunits.

Visceral leishmaniasis (VL) is a fatal infectious disease caused by viscerotropic parasitic species of Leishmania. Current treatment options are often ineffective and toxic, and more importantly, there are no clinically validated drug targets available to develop next generation therapeutics against VL. Topoisomerase IB (TopIB) is an essential enzyme for Leishmania survival. The enzyme is organized as a bi-subunit that is distinct from the monomeric topoisomerase I of human. Based on this unique feature, we synthesized peptides composed of partial amino acid sequences of small subunit of Leishmania donovani (Ld) TopIB to confirm a decrease in catalytic activity by interfering the interaction between the two subunits. One of the synthetic peptides, covering essential amino acids for catalytic activity of LdTopIB, interrupted the enzymatic activity. Next, we examined 151 compounds selected from virtual screening in a functional assay and identified three LRL-TP compounds with a significant decrease in LdTopIB activity (IC50 of LRL-TP-85: 1.3 µM; LRL-TP-94: 2.9 µM; and LRL-TP-101: 35.3 µM) and no effects on Homo sapiens (Hs) TopIB activity. Based on molecular docking, the protonated tertiary amine of inhibitors formed key interactions with S415 of the large subunit. The EC50 values of LRL-TP-85, LRL-TP-94, and LRL-TP-101 were respectively 4.9, 1.4, and 27.8 µM in extracellular promastigote assay and 34.0, 53.7, and 11.4 µM in intracellular amastigote assay. Overall, we validated the protein-protein interaction site of LdTopIB as a potential drug target and identified small molecule inhibitors with anti-leishmanial activity.

Pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs as possible candidates against neglected tropical diseases (NTDs): identification of hit compounds towards development of potential treatment of Leishmania donovani.

A rational-based process was adopted for repurposing pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs bearing variable acyl chains, different stereochemical configuration and/or positional relationships. Structural features were highly influential on activity. Amongst, enantiomer 1e having 1,2-vicinal relationship for the -CH2O- and the N-acyl moieties, a saturated palmitoyl chain and an opposite stereochemical configuration to natural sphingolipids was the most potent hit compound against promastigotes showing IC50 value of 28.32 µM. The corresponding enantiomer 1a was 2-fold less potent showing a eudismic ratio of 0.54 in promastigotes. Compounds 1a and 1e inhibited the growth of amastigotes more potently relative to promastigotes. Amongst, enantiomer 1a as the more selective and safer. In silico docking study using a homology model of Leishmania donovani inositol phosphoceramide synthase (IPCS) provided plausible reasoning for the molecular factors underlying the found activity. Collectively, this study suggests compounds 1a and 1e as potential hit compounds for further development of new antileishmanial agents.

In Vitro and in Vivo Activity of mTOR Kinase and PI3K Inhibitors Against Leishmania donovani and Trypanosoma brucei.

Kinetoplastid parasites, including Leishmania and Trypanosoma spp., are life threatening pathogens with a worldwide distribution. Next-generation therapeutics for treatment are needed as current treatments have limitations, such as toxicity and drug resistance. In this study, we examined the activities of established mammalian target of rapamycin (mTOR)/phosphoinositide 3-kinase (PI3K) inhibitors against these tropical diseases. High-throughput screening of a library of 1742 bioactive compounds against intracellular L. donovani was performed, and seven mTOR/PI3K inhibitors were identified. Dose-dilution assays revealed that these inhibitors had half maximal effective concentration (EC50) values ranging from 0.14 to 13.44 µM for L. donovani amastigotes and from 0.00005 to 8.16 µM for T. brucei. The results of a visceral leishmaniasis mouse model indicated that treatment with Torin2, dactolisib, or NVP-BGT226 resulted in reductions of 35%, 53%, and 54%, respectively, in the numbers of liver parasites. In an acute T. brucei mouse model using NVP-BGT226 parasite numbers were reduced to under the limits of detection by five consecutive days of treatment. Multiple sequence and structural alignment results indicated high similarities between mTOR and kinetoplastid TORs; the inhibitors are predicted to bind in a similar manner. Taken together, these results indicated that the TOR pathways of parasites have potential for the discovery of novel targets and new potent inhibitors.

Defective Ca(2+) binding in a conserved binding site causes incomplete N-glycan processing and endoplasmic reticulum trapping of discoidin domain receptors.

An X-ray crystallographic study has suggested that vertebrate discoidin domain receptors (DDRs) have a conserved Ca(2+) binding site. DDR1 and DDR2 transfected in HEK293 cells were expressed mainly as 120 and 130 kDa forms, respectively, as they are sufficiently N-glycosylated. However, both of them showed the molecular weight of 110 kDa predominantly in the cells cultured with Ca(2+)-depleted media. DDR2-carrying D234A mutation at the conserved Ca(2+)-binding site expressed the 110 kDa form dominantly even in normal culture condition. DDR2 becomes 100 kDa form in glucose-depleted culture condition and its molecular weight increases up to 130 kDa with re-feeding glucose. However, in the mutant DDR2, the increase came to a halt at 110 kDa. The 110 kDa form had premature N-glycosyl carbohydrates and located predominantly within the endoplasmic reticulum. These results suggest that DDRs require Ca(2+)-binding to complete their N-glycan processing and generate the form targeted to cell membrane.

Topical application of ALK5 inhibitor A-83-01 reduces burn wound contraction in rats by suppressing myofibroblast population.

Burn scar contracture that follows the healing of deep dermal burns causes severe deformation and functional impairment. However, its current therapeutic interventions are limited with unsatisfactory outcomes. When we treated deep second-degree burns in rat skin with activin-like kinase 5 (ALK5) inhibitor A-83-01, it reduced wound contraction and enhanced the area of re-epithelialization so that the overall time for wound closing was not altered. In addition, it reduced myofibroblast population in the dermis of burn scar with a diminished deposition of its biomarker proteins such as α-SMA and collagen. Treatment of rat dermal fibroblast with A-83-01 inhibited transforming growth factor-ß1 (TGF-ß1)-dependent induction of α-SMA and collagen type I. Taken together, these results suggest that topical application of ALK5 inhibitor A-83-01 could be effective in preventing the contraction of burn wound without delaying the wound closure by virtue of its inhibitory activity against the TGF-ß-induced increase of myofibroblast population.

Identification of Novel Flavonoids and Ansa-Macrolides with Activities against Leishmania donovani through Natural Product Library Screening.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis (VL), a potentially fatal disease if left untreated. Given the limitations of current therapies, there is an urgent need for new, safe, and effective drugs. To discover novel antileishmanial compounds from previously unexplored chemical spaces, we conducted a high-throughput screening (HTS) of 2562 natural compounds, assessing their activity against L. donovani promastigotes and intracellular amastigotes. Utilizing the criteria of ≥70% parasite growth inhibition and ≥70% host cell (THP-1) viability, we selected 100 inhibitors for half-maximal inhibitory concentration (IC50) value determination. Twenty-six compounds showed activities in both forms of Leishmania with a selectivity index of over 3. Clustering analysis resulted in four chemical clusters with scaffolds of lycorine (cluster 1), 5-hydroxy-9,10-dihydro-4H,8H-pyrano[2,3-f]chromene-4,8-dione (cluster 2), and semi-synthetic derivatives of ansamycin macrolide (cluster 4). The enantiomer of lycorine, BMD-NP-00820, showed the highest anti-amastigote activity with an IC50 value of 1.74 ± 0.27 µM and a selectivity index (SI) > 29. In cluster 3, the most potent compound had an IC50 value of 2.20 ± 0.29 µM with an SI > 23, whereas in cluster 4, with compounds structurally similar to the tuberculosis drug rifapentine, BMD-NP-02085 had an IC50 value of 1.76 ± 0.28 µM, but the SI value was 7.5. Taken together, the natural products identified from this study are a potential source for the discovery of antileishmanial chemotypes for further development.

Low stability and a conserved N-glycosylation site are associated with regulation of the discoidin domain receptor family by glucose via post-translational N-glycosylation.

Cell-surface expression of the discoidin domain receptor (DDR) tyrosine kinase family in high molecular mass form was controlled sensitively by the glucose concentration through a post-translational N-glycosylation process. Cycloheximide time-course experiments revealed that the high-molecular-mass forms of DDR1 and DDR2 were significantly less stable than control receptor tyrosine kinases. Site-directed mutational analysis of the consensus N-glycosylation sites of the DDRs revealed that mutations of asparagine 213 of DDR2 and asparagine 211 of DDR1, a conserved N-glycosylation site among vertebrate DDRs, inhibited the generation of the high-molecular-mass isoform. Taken together, these results suggest a mechanism to control the activity of the DDR family by regulating its cell-surface expression. Due to low stability, the steady-state population of functional DDR proteins in the cell surface depends sensitively on its maturation process via post-translational N-glycosylation, which is controlled by the glucose supply and the presence of a conserved N-glycosylation site.

Design, synthesis, and repurposing of O6-aminoalkyl-sulfuretin analogs towards discovery of potential lead compounds as antileishmanial agents.

Up to date, there are still significantly unmet clinical needs for treatment of the fatal visceral leishmaniasis; a neglected tropical disease. Herein, a recently reported antileishmanial hit sulfuretin analog suffering from a low potency and a problematic aqueous solubility that hindered further development was used as a starting point. A mitigation rational via incorporation of O6-aminoalkyl moiety suggest structures analogous to literature-known compounds as cholinesterase inhibitors. Consequently, preparation and repurposing of a library of these compounds unveiled their potential activity against the parasite Leishmania donovani promastigotes. Further evaluation against intracellular form of the parasite and host cells suggested compounds 2a, 2c, and 2o derived from sulfuretin analogs bearing 2'-methoxy or 2',5'-dimethoxy substituents at ring-B as promising lead compounds with potential activity and acceptable safety window relative to the standard edelfosine. In silico simulation predicted plausible binding modes of these compounds to L. donovani fumarate reductase. Together this work presents compound 2o as a potential lead compound for further development.

Design, Synthesis, and Repurposing of Rosmarinic Acid-ß-Amino-α-Ketoamide Hybrids as Antileishmanial Agents.

A series of rosmarinic acid-ß-amino-α-ketoamide hybrids were synthesized and rationally repurposed towards the identification of new antileishmanial hit compounds. Two hybrids, 2g and 2h, showed promising activity (IC50 values of 9.5 and 8.8 µM against Leishmania donovani promastigotes, respectively). Their activities were comparable to erufosine. In addition, cytotoxicity evaluation employing human THP-1 cells revealed that the two hybrids 2g and 2h possess no cytotoxic effects up to 100 µM, while erufosine possessed cytotoxicity with CC50 value of 19.4 µM. In silico docking provided insights into structure-activity relationship emphasizing the importance of the aliphatic chain at the α-carbon of the cinnamoyl carbonyl group establishing favorable binding interactions with LdCALP and LARG in both hybrids 2g and 2h. In light of these findings, hybrids 2g and 2h are suggested as potential safe antileishmanial hit compounds for further development of anti-leishmanial agents.

Bestatin analogs-4-quinolinone hybrids as antileishmanial hits: Design, repurposing rational, synthesis, in vitro and in silico studies.

Amongst different forms of leishmaniasis, visceral leishmaniasis caused by L. donovani is highly mortal. Identification of new hit compounds might afford new starting points to develop novel therapeutics. In this lieu, a rationally designed small library of bestatin analogs-4-quinolone hybrids were prepared and evaluated. Analysis of SAR unveiled distinct profiles for hybrids type 1 and type 2, which might arise from their different molecular targets. Amongst type 1 bestatin analog-4-quinolone hybrids, hybrid 1e was identified as potential hit inhibiting growth of L. donovani promastigotes by 91 and 53% at 50 and 25 µM concentrations, respectively. Meanwhile, hybrid 2j was identified amongst type 2 bestatin analog-4-quinolone hybrids as potential hit compound inhibiting growth of L. donovani promastigotes by 50 and 38% at 50 and 25 µM concentrations, respectively. Preliminary safety evaluation of the promising hit compounds showed that they are 50-100 folds safer against human derived monocytic THP-1 cells relative to the drug erufosine. In silico study was conducted to predict the possible binding of hybrid 1e with methionine aminopeptidases 1 and 2 of L. donovani. Molecular dynamic simulations verified the predicted binding modes and provide more in depth understanding of the impact of hybrid 1e on LdMetAP-1 and LdMetAP-2.

Synthesis and Testing of Analogs of the Tuberculosis Drug Candidate SQ109 against Bacteria and Protozoa: Identification of Lead Compounds against Mycobacterium abscessus and Malaria Parasites.

SQ109 is a tuberculosis drug candidate that has high potency against Mycobacterium tuberculosis and is thought to function at least in part by blocking cell wall biosynthesis by inhibiting the MmpL3 transporter. It also has activity against bacteria and protozoan parasites that lack MmpL3, where it can act as an uncoupler, targeting lipid membranes and Ca2+ homeostasis. Here, we synthesized 18 analogs of SQ109 and tested them against M. smegmatis, M. tuberculosis, M. abscessus, Bacillus subtilis, and Escherichia coli, as well as against the protozoan parasites Trypanosoma brucei, T. cruzi, Leishmania donovani, L. mexicana, and Plasmodium falciparum. Activity against the mycobacteria was generally less than with SQ109 and was reduced by increasing the size of the alkyl adduct, but two analogs were ∼4-8-fold more active than SQ109 against M. abscessus, including a highly drug-resistant strain harboring an A309P mutation in MmpL3. There was also better activity than found with SQ109 with other bacteria and protozoa. Of particular interest, we found that the adamantyl C-2 ethyl, butyl, phenyl, and benzyl analogs had 4-10× increased activity against P. falciparum asexual blood stages, together with low toxicity to a human HepG2 cell line, making them of interest as new antimalarial drug leads. We also used surface plasmon resonance to investigate the binding of inhibitors to MmpL3 and differential scanning calorimetry to investigate binding to lipid membranes. There was no correlation between MmpL3 binding and M. tuberculosis or M. smegmatis cell activity, suggesting that MmpL3 is not a major target in mycobacteria. However, some of the more active species decreased lipid phase transition temperatures, indicating increased accumulation in membranes, which is expected to lead to enhanced uncoupler activity.

LCB 03-0110, a novel pan-discoidin domain receptor/c-Src family tyrosine kinase inhibitor, suppresses scar formation by inhibiting fibroblast and macrophage activation.

Wound healing generally induces an inflammatory response associated with tissue fibrosis in which activated macrophage and myofibroblast cells are primarily involved. Although this is known to be the underlying mechanism for scarring and various fibrotic pathologies, no effective intervention is currently available. We identified (3-(2-(3-(morpholinomethyl)phenyl)thieno[3,2-b]pyridin-7-ylamino)phenol (LCB 03-0110), a thienopyridine derivative, as a potent inhibitor of discoidin domain receptor family tyrosine kinases and discovered that this compound strongly inhibits several tyrosine kinases, including the c-Src family, spleen tyrosine kinase, Bruton's tyrosine kinase, and vascular endothelial growth factor receptor 2, which are important for immune cell signaling and inflammatory reactions. LCB 03-0110 suppressed the proliferation and migration of primary dermal fibroblasts induced by transforming growth factor ß1 and type I collagen, and this result correlated with the inhibition ability of the compound against enhanced expression of α-smooth muscle actin and activation of Akt1 and focal adhesion kinase. In J774A.1 macrophage cells activated by lipopolysaccharide LCB 03-0110 inhibited cell migration and nitric oxide, inducible nitric-oxide synthase, cyclooxygenase 2, and tumor necrosis factor-α synthesis. LCB 03-0110 applied topically to full excisional wounds on rabbit ears suppressed the accumulation of myofibroblast and macrophage cells in the healing wound and reduced hypertrophic scar formation after wound closing, without delaying the wound closing process. Taken together, the pharmacological activities of LCB 03-0110 suggest that it could be an effective agent for suppressing fibroinflammation by simultaneously targeting activated fibroblasts and macrophages.

Design, Rational Repurposing, Synthesis, In Vitro Evaluation, Homology Modeling and In Silico Study of Sulfuretin Analogs as Potential Antileishmanial Hit Compounds.

Direct growth inhibition of infectious organisms coupled with immunomodulation to counteract the immunosuppressive environment might be a beneficial therapeutic approach. Herein, a library of sulfuretin analogs were developed with potential capabilities to inhibit production of the immunosuppressive PGE2 and elicit direct growth inhibition against Leishmania donovani; the major causative agent of the fatal visceral leishmaniasis. Amongst explored library members bearing diverse methoxy and/or hydroxy substitution patterns at rings B and A, analog 1i retaining the C6-hydroxy moiety at ring-A, but possessing methoxy moieties in place of the polar dihydroxy moieties of sulfuretin ring-B, as well as analog 1q retaining the sulfuretin's polar dihydroxy moieties at ring-B, but incorporating a C6-methoxy moiety instead of the C6-hydroxy moiety at ring-A, were the most promising hit compounds. Cytotoxicity evaluation suggested that analog 1i possesses a safety profile inducing the death of the parasite rather than host cells. In silico simulation provided insights into their possible binding with Leishmania donovani fumarate reductase. The current investigation presents sulfuretin analogs 1i and 1q as potential hit compounds for further development of multifunctional therapeutic agents against visceral leishmaniasis.

In Vivo Efficacy of SQ109 against Leishmania donovani, Trypanosoma spp. and Toxoplasma gondii and In Vitro Activity of SQ109 Metabolites.

SQ109 is an anti-tubercular drug candidate that has completed Phase IIb/III clinical trials for tuberculosis and has also been shown to exhibit potent in vitro efficacy against protozoan parasites including Leishmania and Trypanosoma cruzi spp. However, its in vivo efficacy against protozoa has not been reported. Here, we evaluated the activity of SQ109 in mouse models of Leishmania, Trypanosoma spp. as well as Toxoplasma infection. In the T. cruzi mouse model, 80% of SQ109-treated mice survived at 40 days post-infection. Even though SQ109 did not cure all mice, these results are of interest since they provide a basis for future testing of combination therapies with the azole posaconazole, which acts synergistically with SQ109 in vitro. We also found that SQ109 inhibited the growth of Toxoplasma gondii in vitro with an IC50 of 1.82 µM and there was an 80% survival in mice treated with SQ109, whereas all untreated animals died 10 days post-infection. Results with Trypanosoma brucei and Leishmania donovani infected mice were not promising with only moderate efficacy. Since SQ109 is known to be extensively metabolized in animals, we investigated the activity in vitro of SQ109 metabolites. Among 16 metabolites, six mono-oxygenated forms were found active across the tested protozoan parasites, and there was a ~6× average decrease in activity of the metabolites as compared to SQ109 which is smaller than the ~25× found with mycobacteria.

Characterization of Methylophaga sp. strain SK1 cytochrome cL expressed in Escherichia coli.

Methylophaga sp. strain SK1 is a new restricted facultative methanol-oxidizing bacterium that was isolated from seawater. The aim of this study was to characterize the electron carriers involved in the methanol oxidation process in Methylophaga sp. strain SK1. The gene encoding cytochrome c(L) (mxaG) was cloned and the recombinant gene was expressed in Escherichia coli DH5 under strict anaerobic conditions. The recombinant cytochrome c(L) had the same molecular weight and absorption spectra as the wild-type cytochrome c(L) both in the reduced and oxidized forms. The electron flow rate from methanol dehydrogenase (MDH) to the recombinant cytochrome c(L) was similar to that from MDH to the wild-type cytochrome c(L). These results suggest that recombinant cytochrome c(L) acts as a physiological primary electron acceptor for MDH.