We explore a novel concept of metal Chelation-assisted Solubility Enhancement (CHASE). This study is based on complexation with Na+ or K+. A series of 6 compounds are studied by conjugating a hydrophobic moiety with benzo-15-crown-5. LogP values are determined in the absence and presence of Na+ or K+, respectively. The largest shift upon metal chelation is with compound 7 (a pyrenyl-crown ether conjugate), which has a logP of 3.61 in the absence of any metal ion and 0.99 and 1.45 in the presence of Na+ or K+, respectively. Such results clearly demonstrate the feasibility of the proposed approach. Beyond chelation, cation-π interactions seem to play a substantial role in determining the partitioning outcome for those with an aryl system. Such findings suggest the need for all future partition experiments to incorporate biologically relevant concentrations of metal ions. This feasibility study opens a new direction in improving drug solubility.

Machupo virus (MACV) is the causal agent of Bolivian Hemorrhagic fever. It is highly pathogenic, has a high mortality rate, and currently lacks specific treatments or vaccines. MACV belongs to the Arenaviridae family, which uses a cap-snatching mechanism during the transcription process. Its viral polymerase, the L protein, harbors the endonuclease activity required for cap snatching, making it a suitable target for the development of antiviral therapeutics. We combined experimental and computational methods to characterize MACV endonuclease activity and evaluate inhibitors. A fluorescence resonance energy transfer (FRET) assay was used to measure the enzymatic activity of endonuclease and identify potent inhibitors via high-throughput screening. FRET assays identified BW-148, an inhibitor with a 48.4 µM (95% CI: 37.3–59.3 µM; R2 = 0.98) IC50, and a KD of 13.7 µM (95% CI: 8.2–19.2 µM, n = 3). Docking studies reveal that BW-148 may bind near the MACV endonuclease catalytic site, inhibiting enzymatic activities by metal chelating. BW-148 is a useful lead compound for further optimization of Machupo endonuclease inhibitors.

Redox homeostasis is a critical part of physiological processes. Disruption of such homeostasis is associated with various pathological conditions. Tools for studying cellular redox states are important for understanding various biological mechanisms and for the targeted delivery of drugs and/or imaging agents. Along this line, there have been reaction-based fluorescent probes capable of detecting either oxidative stress or hypoxia. However, tools are not available to detect dynamic redox changes in either direction. Toward that end, we describe chemistry that allows for detecting both reduction and oxidation changes using a probe redox pair, moving toward monitoring dynamic redox processes. Embedded in this new approach is also new chemistry for fluorophore activation through the selective reduction of a sulfone group to a thioether under severe hypoxia, complementing the widely used nitro or N-oxide reduction approaches. We demonstrate the feasibility through solution-phase studies and cell-culture work in two cell lines: cancer cells and macrophages. One optimized redox probe pair was studied for its response to both induction of highly reactive oxygen species (hROS) production by turning off the fluorescence and severe hypoxia with fluorescence turn-on. Overall, this approach sets a new direction in designing redox-sensitive tools.

Reports of carbon monoxide (CO) pharmacology have spurred intense interest in developing its fluorescent probes with much success. However, one unfortunate event in this area is the wide-spread use of chemically reactive metal/BH3-CO complexes as “CO-releasing molecules” or CORMs that do not produce CO or produce CO in an idiosyncratic fashion. Consequently, a large number of reported fluorescent “CO probes” only respond to the CORM used, but not to CO. Though most of these issues have been clarified in the literature, there is a surprising recent publication on a Cu(II)-based fluorescent “CO probe,” Nap-BC-Cu(II), relying on undefined chemical principles. We reassessed the ability for Nap-BC-Cu(II) to detect CO and found no evidence for Nap-BC-Cu(II) to selectively detect CO at even non-physiologically relevant high concentrations (high micromolar) of CO. Marginal effects were observed only when CO was continuously bubbled through the “probe” solution for 15 min. Further, Nap-BC-Cu(II) was found to be sensitive to ascorbic acid and cysteine. Overall, this probe did not respond to CO in a pathophysiologically relevant context. Our findings do not support the notion of Nap-BC-Cu(II) being a CO probe for studying CO biology. We hope this will be the last of this saga of “CO probes” that do not afford selective detection of CO, largely due to the confusions caused by using chemically reactive CORMs.

In studying the roles of reactive oxygen species (ROS) in various biological processes, the availability of appropriate cell culture models is critical. Addition of H2O2 to cells is commonly used to simulate oxidative stress. In doing so, generation of highly reactive oxygen species (hROS) in cell culture is used as an indication of successful model creation. The validity of such a model is predicated on the assumption that hROS formation is the result of cellular biochemical processes and not from the medium. However, we observed a significant level of hROS in various culture media alone upon H2O2 addition, raising questions about the validity of such models and suggesting a “Trojan Horse” role for such media in compromising the data. Given the wide-spread use of the said method, we urge caution in analysing information gained from such models in redox mechanistic studies.

Because of the central role of the mitochondrion in various pathophysiological processes, the ability to target therapeutic and imaging agents to the mitochondrion has immense application potential. The most commonly used targeting moiety is triphenyl phosphine (TPP), which brings in undesirable traits such as high hydrophobicity (log P= 5.7) and inhibition of mitochondrial functions. We are interested in searching for a targeting moiety, which is much smaller than TPP and introduces minimal hydrophobicity and toxicity issues. Herein, we describe our work of studying lutidine as a mitochondrion-targeting moiety. We are interested in lutidine because of its small size, desirable log P (~1.7), and known safety as a food additive. Using LC-MS and fluorescence as cross validating methods, mouse liver mitochondria, and TPP conjugates as positive controls, we demonstrate that lutidine conjugates are able to enrich in the mitochondrion substantially (~250-1000 fold). Even the lower level of enrichment fluctuation offers significant application potential in targeted delivery of drugs and/or imaging agents.

Most ROS-sensitive cleavable linkers rely on their broad reactivity toward all ROS. However, individual ROS have very specialized functions in various pathologies. For example, HOCl/OCl- is primarily produced in response to infection and/or inflammation by certain immune cells that express myeloperoxidase (MPO). We herein describe a novel HOCl/OCl--selective prodrug approach through an oxidation-initiated Cascade Reaction with Kinetic Tunability (CReKT) for drug release. Specifically, HOCl/OCl- oxidation of a phenylthioether is used to trigger prodrug activation via enhancing the nucleophilicity of the S-connected carbon for condensation-based payload release. The reactivity of the S-connected carbon is further augmented by tethering to an electron-withdrawing group (EWG) and by creating synergy with proximity effects. Tunability of release kinetics can be achieved by varying the EWG, substitution on the phenyl ring, and entropic factors. This approach offers new tools and sets a new direction in designing species-selective ROS-sensitive prodrugs.

Metastatic recurrence is the principal cause of cancer mortality. Pancreatic ductal adenocarcinoma (PDAC) and triple-negative breast cancer (TNBC) recur frequently after apparently curative therapy. Carbon monoxide (CO) is an endogenously produced signaling molecule with cytoprotective properties, but clinical use of CO gas is constrained by safety and dose-control challenges. Here, a metal-free CO prodrug (CO-116) suppresses metastatic progression in vivo. In experimental models of PDAC and TNBC, CO-116 reduces metastatic burden without evidence of overt toxicity while maintaining carboxyhemoglobin within physiological ranges. At an equivalent total weekly dose, dividing the dose into more frequent, lower administrations achieves greater efficacy than a single weekly dose, indicating schedule dependence. Mechanistically, CO-116 downregulates the heme importer HRG1 and attenuates a downstream CYP1B1-SP1 program; gain- and loss-of-function studies establish HRG1 as a functional mediator of metastatic progression and CO responsiveness. An independent cohort using a distinct metal-free scaffold (CO-103) likewise reduces metastasis in PDAC, supporting a scaffold-independent class effect. These findings establish a mechanistically anchored, non-inhaled CO strategy to suppress metastasis and motivate adjuvant development focused on schedule optimization and biomarker-guided dosing.

Carbon monoxide (CO) is an endogenous signaling molecule. It is produced via heme degradation by heme oxygenase (HMOX), releasing stoichiometric amounts of CO, iron, and biliverdin (then bilirubin). The HMOX-CO axis has long been shown to offer beneficial effects by modulating inflammation, proliferation and cell death as they relate to tissue and organ protection. Recent years have seen a large number of studies examining CO pharmacology, its molecular targets, cellular mechanisms of action, pharmacokinetics, and detection methods using various delivery modalities including inhaled CO gas, CO solutions, and various types of CO donors. Unfortunately, one widely used donor type includes four commercially available carbonyl complexes with metal or borane, CORM-2 (Ru2+), CORM-3 (Ru2+), CORM-A1 (BH3), and CORM-401 (Mn+), which have been shown to have minimal and/or unpredictable CO production and extensive CO-independent chemical reactivity and biological activity. As a result, not all "CO biological activities" in the literature can be attributed to CO. In this review, we summarize key findings based on CO gas and CO in solution for the certainty of the active principal and to avoid data contamination resulting from the confirmed or potential reactivities and activities of the "carrier" portion of CORMs. Along a similar line, we discuss interesting potential research areas of CO in the brain including a newly proposed CO/HMOX/dopamine axis and the role of CO in cognitive stimulation and circadian rhythm. This review is critical for the future development of the CO field by steering clear of complications caused by chemically reactive donor molecules.

Three new tirucallane-type triterpenes, tirucalla-7,24-diene-1β,3β-diol (oddurensinoid B), tirucalla-7-ene-1β,3β,25-triol (oddurensinoid H), and tirucalla-7, 24-diene-3β-ol-1-O-β-d-glucopyranoside (oddurensinoid K), were isolated from the resin of Commiphora oddurensis harvested from Ethiopia, Africa. Their structures were elucidated by one-dimensional (1D) NMR, two-dimensional (2D) NMR, and high-resolution mass spectrometry (HRMS). All three compounds were tested for their anticancer activity against HeLa cell lines. They all exhibit anticancer activity, with oddurensinoid H the most potent with IC50 of 0.017 mg/mL (36.9 μM).

Because of the increasing interests in carbon monoxide (CO) as an endogenous signaling molecule, there have been extensive efforts in developing fluorescent probes for CO. In doing so, metal-carbonyl complexes named "CO-releasing molecules" (CORMs) are often used as CO surrogates. The most widely used CORM-2 and CORM-3 are chemically reactive Ru(II) complexes; release minimal or no CO unless in the presence of a strong nucleophile or a reducing agent; and do not function as reliable CO donors. As a result, some reported CO fluorescent probes only detect the CORM used, not CO. Recently, an Fe(III)-fluorophore complex, RBF-Fe(III), has been reported to sense CO using CORM-3 as a CO surrogate. The proposed mechanism involves CO binding to Fe(III). Because of the known affinity of CO for only Fe(II), but not Fe(III), we were intrigued by the report. Re-evaluation work found fluorescence changes of RBF-Fe(III) by CORM-3, but not CO itself. Furthermore, sodium ascorbate and cysteine were found to induce fluorescent changes of the RBF-Fe(III) system. Moreover, RBF-Fe(III) was found to be unstable and to change fluorescence with time or agitation. Regardless of whether it was under N2, CO, or vacuum, vigorous stirring induced the same level of fluorescence changes, presumably due to precipitation or aggregation of Fe(III) species, which is consistent with literature findings of Fe(III) behaviors. Such results mean that the RBF-Fe(III) system does not sense CO and underscore the need to exercise extra cautions when chemically reactive CO donors are used in developing CO probes.

Pd(0)-mediated deallylation has been employed for developing fluorescent probes for carbon monoxide (CO). The key idea relied on the ability of CO to reduce Pd(II) to Pd(0). However, most studies used Ru-based CORM-2 and/or CORM-3 as CO sources, despite their known chemical reactivity and idiosyncratic CO release. Herein, we conducted studies using one of the most widely used probes (FL-CO-1), evaluating its response to various CO sources and to Pd(0). We found that (1) the activation of FL-CO-1 by CORM-2/-3 has CO-independent component(s); (2) vitamin C and cysteine were found to interfere with the probe's performance; and (3) Pd(0) only led to moderate fluorescence turn-on, while a combination of Pd(0) and CO resulted in a pronounced fluorescence turn-on response. Such findings indicate that the role(s) of CO goes beyond Pd(II) reduction, and accurate in vivo detection of CO using this approach is unlikely because of the presence of vitamin C and thiols in living systems. These new insights suggest the need to reinterpret some results, particularly when chemically reactive CORM-2 and CORM-3 were employed as CO surrogates. We recommend that future studies avoid using reactive CORMs to ensure experimental rigor.

This study reports the isolation of the known triterpenoid myrrhasin (1) from the EtOAc/MeOH (1:1, v/v) extract of Commiphora oddurensis Chiov. resin, occurring in the Ogaden region of Ethiopia, along with its structural elucidation. Notably, this is only the second report of myrrhasin's occurrence in nature, previously described by Tao Shen et al. from C. myrrha, though the source material is questionable. Nevertheless, our examination of authentic myrrh demonstrates that myrrhasin is absent from C. myrrha gum resin, recognizing a pivotal discovery as no triterpene compound has been reported from this species until now. This finding is crucial given the cultural, commercial, and medicinal importance of myrrh. This study also provides a brief description of the functional group modification of myrrhasin (1) that results in its epoxide (1a/b) and acetate (1c) derivatives. Furthermore, anticancer activity tests of the crude extract, compound 1, and derivatives 1a/b on cervical cancer cells (HeLa cells) exhibited IC50 values of 33, 29, and 35 µg/mL, respectively, highlighting their potential therapeutic applications.

Targeted delivery of carbon monoxide (CO) prodrugs holds important therapeutic potential for various applications. Along this line, we developed an enrichment-triggered release (ETR) approach for activating 2-component (a diene and a dienophile) CO prodrugs upon enrichment in the mitochondrion, giving a "one stone, two birds" approach. Herein, we aim to broaden the scope of application to targeted delivery to the lysosome. We tethered a CO prodrug pair, a diene and a dienophile, with morpholine, a lysosomal targeting moiety. Several analogs were synthesized to tune the second-order rate constants (k2) to a desirable range. We chose two pairs of the prodrugs with different second-order rate constants (0.087 and 0.21 M-1 s-1) to further study their enrichment and CO release ability. For one pair, LC-MS experiments revealed > 13-fold enrichment of the morpholine-conjugated CO prodrug pair compared to non-targeted controls in HeLa cells. Fluorescence studies demonstrated the same enrichment and co-localization of LysoTracker. For the second pair, conjugation with morpholine did not lead to improved enrichment in the lysosome. This study represents the first demonstration of lysosome-targeted delivery of CO. However, our findings also note the nonuniversal nature for a morpholine moiety to lead to lysosomal enrichment. The modest magnitude of enrichment also means that this method may only be applicable for targeted delivery of a highly potent drug.

Carbon monoxide (CO) is an endogenously produced gaseous signaling molecule that has been shown to have therapeutic values. In studying CO pharmacology, dose dependency has not been properly studied in most, if not all, such work. Part of the reason is the difficulty in determining the solution concentration of a gaseous molecule (CO) with limited water solubility ( 1 mM). Along this line, CO solution prepared at a pre-determined concentration has been widely used in studying CO pharmacology. However, different from making a solution of a non-volatile small-molecule drug, CO is expected to quickly escape from the solution, leading to unknown concentrations and an intractable scenario for dose-dependency studies. In this study, we hope to help define the boundary conditions by studying the concentration decay profiles of CO in solution at a pre-determined concentration. Results from such studies will be very important foundational information for future dose-dependency studies of CO pharmacology.

Inflammation is a pathology implicated in a wide range of human diseases. Recent years have seen tremendous progress in developing new types of anti-inflammatory agents for the treatment of inflammation of various origins. However, each has its own strengths and weaknesses. The very fact that there needs to have multiple types of anti-inflammatory agents underlines the complexity of inflammatory diseases and conditions, their molecular origins, and their treatment. Such complexity dictates the need to search for new approaches with improved potency and efficacy as well as reduced side effects. For these reasons, we are interested in exploring the possibility of generating synergy between carbon monoxide (CO), an endogenously produced cytoprotective agent, and known anti-inflammatory agents. Herein, we report the potentiating actions of CO on the anti-inflammatory effects of cortisone and dexamethasone as demonstrated in their ability to suppress the expression of TNF-α and IL-6 induced by either LPS or the S protein of SARS-CoV-2. Such effects are reflected in the substantially increased potency as well efficacy, when the efficacy of the corticosteroid alone does not allow for complete suppression of the expression of these cytokines. Further, increased attenuation of p65 phosphorylation is at least part of the molecular mechanism for the observed potentiating effects. We hope our work will stimulate a high level of activity along the same direction, leading to anti-inflammatory strategies with improved potency and efficacy and reduced side effects.

The Trypanosoma brucei group of parasites causes Nagana in cattle and human African trypanosomiasis, or sleeping sickness, in humans. Current drugs against these parasites have severe toxicity, vaccines are not available, and development of drug resistance makes finding new chemotherapeutic targets imperative. Ion channels, which are involved in several biological processes, are targets of many therapeutically useful agents, and they remain significantly underexplored as therapeutic targets in parasites. Here, we report the presence of a voltage gated Ca2+ channel (VGCC, TbCav), which is localized in the flagellar plasma membrane (PM) of T. brucei and is essential for proliferation of both bloodstream (BSF) and procyclic forms (PCF) of the parasite. TbCaV is a single subunit channel capable of transporting Ca2+ when expressed in mutant yeast lacking PM Ca2+ channels or in HEK293T cells. Through the virtual screening of a commercial chemical library using dynamic ensembles of various conformations of TbCav and associated docking analyses, several inhibitors of TbCav were discovered. As pharmacological validation of the essential roles of TbCav, these compounds were shown to inhibit T. brucei growth with the most potent agent, N-(7-nitro-2,1,3-benzoxadiazol-4-yl) acetamide (NBD-A), exhibiting an EC50 of 25 ± 3 nM and no cytotoxicity in Vero cells possessing related channels. Thus, such studies constitute pharmacological validation of TbCav as a viable therapeutic target of T. brucei.

Sodium-dependent multivitamin transporter (SMVT) is a biotin transporter over-expressed in various types of cancer cells and is commonly studied for targeted drug delivery using biotin conjugates. However, such conjugates lack the carboxyl group needed for recognition by SMVT. Previously, we proposed that SMVT is unlikely the transporter of biotin conjugates. To experimentally assess this hypothesis, we examined intracellular enrichment and activation of the biotin-conjugated version of a well-established CO prodrug pair in cell culture. Although prodrug enrichment in SMVT-over-expressing cells was observed, this enrichment was not affected by excess biotin, indicating the lack of competition for SMVT. Additionally, two biotin analogs lacking the carboxyl group exhibited either augmentative or inhibitory effects depending on specific structural features. These findings support the notion that SMVT is not the transporter of biotin conjugates and underscore the need for further mechanistic studies of the transport mechanism(s) of biotin conjugates.