Modulation of endogenous opioid signaling by inhibitors of puromycin sensitive aminopeptidase.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

Cancer sensitizing effect of deazaflavin analogs is associated with increased intracellular drug accumulation.

As part of our efforts geared towards developing mechanism-based cancer sensitizing agents, we have previously synthesized and characterized novel deazaflavin analogs as potent tyrosyl DNA phosphodiesterase 2 (TDP2) inhibitors for combination treatments with topoisomerase II (TOP2) poisons. Interestingly, the sensitizing effect of a few analogs toward TOP2 poison etoposide (ETP) was associated with a significant increase in intracellular drug accumulation, which could be an alternative mechanism to boost the clinical efficacy of ETP in cancer chemotherapies. Hence, we evaluated more deazaflavin TDP2 inhibitors for their impact on drug retention in cancer cells. We found that all but one tested TDP2 inhibitors substantially increased the ETP retention in DT40 cells. Particularly, we identified an exceptionally potent analog, ZW-1226, which at 3 nM increased the intracellular ETP by 13-fold. Significantly, ZW-1226 also stimulated cellular accumulation of two other anticancer drugs, TOP2 poison teniposide and antifolate pemetrexed, and produced an effect more pronounced than those of ABC transporter inhibitors verapamil and elacridar in human leukemic CCRF-CEM cells toward ETP. Lastly, ZW-1226 potentiated the action of ETP in the sensitive human CCRF-CEM cells and a few resistant non-small-cell lung cancer (NSCLC) cells, including H460 and H838 cells. Collectively, the results of this study strongly suggest that deazaflavin analog ZW-1226 could be an effective cancer sensitizing agent which warrants further investigation.

Enhancing the Antiviral Potency of Nucleobases for Potential Broad-Spectrum Antiviral Therapies.

Broad-spectrum antiviral therapies hold promise as a first-line defense against emerging viruses by blunting illness severity and spread until vaccines and virus-specific antivirals are developed. The nucleobase favipiravir, often discussed as a broad-spectrum inhibitor, was not effective in recent clinical trials involving patients infected with Ebola virus or SARS-CoV-2. A drawback of favipiravir use is its rapid clearance before conversion to its active nucleoside-5'-triphosphate form. In this work, we report a synergistic reduction of flavivirus (dengue, Zika), orthomyxovirus (influenza A), and coronavirus (HCoV-OC43 and SARS-CoV-2) replication when the nucleobases favipiravir or T-1105 were combined with the antimetabolite 6-methylmercaptopurine riboside (6MMPr). The 6MMPr/T-1105 combination increased the C-U and G-A mutation frequency compared to treatment with T-1105 or 6MMPr alone. A further analysis revealed that the 6MMPr/T-1105 co-treatment reduced cellular purine nucleotide triphosphate synthesis and increased conversion of the antiviral nucleobase to its nucleoside-5'-monophosphate, -diphosphate, and -triphosphate forms. The 6MMPr co-treatment specifically increased production of the active antiviral form of the nucleobases (but not corresponding nucleosides) while also reducing levels of competing cellular NTPs to produce the synergistic effect. This in-depth work establishes a foundation for development of small molecules as possible co-treatments with nucleobases like favipiravir in response to emerging RNA virus infections.

Can remdesivir and its parent nucleoside GS-441524 be potential oral drugs? An in vitro and in vivo DMPK assessment.

Remdesivir (RDV) is the only US Food and Drug Administration (FDA)-approved drug for treating COVID-19. However, RDV can only be given by intravenous route, and there is a pressing medical need for oral antivirals. Significant evidence suggests that the role of the parent nucleoside GS-441524 in the clinical outcomes of RDV could be largely underestimated. We performed an in vitro and in vivo drug metabolism and pharmacokinetics (DMPK) assessment to examine the potential of RDV, and particularly GS-441524, as oral drugs. In our in vitro assessments, RDV exhibited prohibitively low stability in human liver microsomes (HLMs, t 1/2 = âˆ¼1 min), with the primary CYP-mediated metabolism being the mono-oxidation likely on the phosphoramidate moiety. This observation is poorly aligned with any potential oral use of RDV, though in the presence of cobicistat, the microsomal stability was drastically boosted to the level observed without enzyme cofactor NADPH. Conversely, GS-441524 showed excellent metabolic stability in human plasma and HLMs. In further in vivo studies in CD-1 mice, GS-441524 displayed a favorable oral bioavailability of 57%. Importantly, GS-441524 produced adequate drug exposure in the mice plasma and lung, and was effectively converted to the active triphosphate, suggesting that it could be a promising oral antiviral drug for treating COVID-19.

Guanabenz Attenuates Acetaminophen-Induced Liver Toxicity and Synergizes Analgesia in Mice.

Endoplasmic reticulum (ER) stress has been shown to be involved in the hepatotoxicity of acetaminophen (APAP). Guanabenz (GA), a widely known antihypertensive drug, is reported to exhibit an anti-ER stress effect. In this study, we investigated the potential of GA as an antidote against APAP-induced hepatotoxicity. The underlying biochemical mechanisms for the hepatoprotective effect of GA were explored. Here we found that treatment of mice with GA (10 mg/kg) before APAP overdose dramatically prevented APAP-induced liver enzyme elevation and resultant toxicity in mice, as indicated by suppression of elevated serum alanine aminotransferase (ALT) levels and liver histological analysis. Importantly, delayed administration of GA within 6 h after APAP overdose also showed an almost equivalent protective effect against APAP liver toxicity. Mechanistically, several pathways are involved in the protective effect of GA against APAP-induced live toxicity, including attenuation of ER stress and oxidative stress, increased levels of nontoxic phase I and II metabolites of APAP, decrease in the formation of toxic N-acetyl-p-benzoquinone imine (NAPQI), and its subsequent protein binding. Importantly, combination of GA with APAP exhibited synergistic interaction in the latter's analgesic activity, while sparing its antipyretic action. These findings provide the preclinical evidence of GA as a promising antidote for treatment of APAP-induced liver toxicity and raise a possibility of its combination with APAP in clinical settings.

Novel deazaflavin tyrosyl-DNA phosphodiesterase 2 (TDP2) inhibitors.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a DNA repair enzyme that removes 5'-phosphotyrosyl blockages resulting from topoisomerase II (TOP2)-DNA cleavage complexes trapped by TOP2 inhibitors. TDP2 is a logical target for the development of therapeutics to complement existing treatments based on inhibition of TOP2. There is, however, no TDP2 inhibitor in clinical development at present. Of the reported TDP2 inhibitors, the deazaflavins are the most promising chemical class centered around the lead compound SV-5-153. Recently we reported new subtypes derived within the deazaflavin family with improved membrane permeability properties. In this work we characterize two representative analogues from two new deazaflavin subtypes based on their biochemical TDP2 inhibitory potency and drug-likeness. We demonstrate that the ZW-1288 derivative represents a promising direction for the development of deazaflavins as therapeutic agents. ZW-1288 exhibits potent inhibitory activity at low nanomolar concentrations against recombinant and cellular human TDP2 with profile similar to that of the parent analog SV-5-153 based on high resistance against murine TDP2 and human TDP2 mutated at residue L313H. While expressing weak cytotoxicity on its own, ZW-1288 potentiates the clinical TOP2 inhibitors etoposide (ETP) and mitoxantrone in human prostate DU145 and CCRF-CEM leukemia and chicken lymphoma DT40 cells while not impacting the activity of the topoisomerase I (TOP1) inhibitor camptothecin or the PARP inhibitor olaparib. ZW-1288 increases the uptake of ETP to a lesser extent than SV-5-153 and remained active in TDP2 knockout cells indicating that the deazaflavin TDP2 inhibitors have additional cellular effects that will have to be taken into account for their further development as TDP2 inhibitors.

Novel Deazaflavin Analogues Potently Inhibited Tyrosyl DNA Phosphodiesterase 2 (TDP2) and Strongly Sensitized Cancer Cells toward Treatment with Topoisomerase II (TOP2) Poison Etoposide.

Topoisomerase II (TOP2) poisons as anticancer drugs work by trapping TOP2 cleavage complexes (TOP2cc) to generate DNA damage. Repair of such damage by tyrosyl DNA phosphodiesterase 2 (TDP2) could render cancer cells resistant to TOP2 poisons. Inhibiting TDP2, thus, represents an attractive mechanism-based chemosensitization approach. Currently known TDP2 inhibitors lack cellular potency and/or permeability. We report herein two novel subtypes of the deazaflavin TDP2 inhibitor core. By introducing an additional phenyl ring to the N-10 phenyl ring (subtype 11) or to the N-3 site of the deazaflavin scaffold (subtype 12), we have generated novel analogues with considerably improved biochemical potency and/or permeability. Importantly, many analogues of both subtypes, particularly compounds 11a, 11e, 12a, 12b, and 12h, exhibited much stronger cancer cell sensitizing effect than the best previous analogue 4a toward the treatment with etoposide, suggesting that these analogues could serve as effective cellular probes.

Discovery of Irreversible p97 Inhibitors.

Inhibitors of human p97 (also known as valosin-containing protein) have been actively pursued because of their potential therapeutic applications in cancer and other diseases. However, covalent and irreversible p97 inhibitors have not been well explored. Herein, we report our design, synthesis, and biological evaluation of covalent and irreversible inhibitors of p97. Among an amide and a reverse amide series we synthesized, we have identified a p97 inhibitor whose functional irreversibility has been established both in vitro and in cells. Also importantly, mass spectrometry reveals three potential cysteine residues labeled by this compound, and mutagenesis together with computer modeling suggests Cys522 as a major site, which when modified, could compromise the function of p97. Taken together, this new inhibitor may provide a template for designing more potent p97 inhibitors with covalent and irreversible characteristics.

Triazolopyrimidine and triazolopyridine scaffolds as TDP2 inhibitors.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) repairs topoisomerase II (TOP2) mediated DNA damages and causes cellular resistance to clinically used TOP2 poisons. Inhibiting TDP2 can potentially sensitize cancer cells toward TOP2 poisons. Commercial compound P10A10, to which the structure was assigned as 7-phenyl triazolopyrimidine analogue 6a, was previously identified as a TDP2 inhibitor hit in our virtual and fluorescence-based biochemical screening campaign. We report herein that the hit validation through resynthesis and structure elucidation revealed the correct structure of P10A10 (Chembridge ID 7236827) to be the 5-phenyl triazolopyrimidine regioisomer 7a. Subsequent structure-activity relationship (SAR) via the synthesis of a total of 47 analogues of both the 5-phenyl triazolopyrimidine scaffold (7) and its bioisosteric triazolopyridine scaffold (17) identified four derivatives (7a, 17a, 17e, and 17z) with significant TDP2 inhibition (IC50 < 50 µM), with 17z showing excellent cell permeability and no cytotoxicity.

5-Aminothiophene-2,4-dicarboxamide analogues as hepatitis B virus capsid assembly effectors.

Chronic hepatitis B virus (HBV) infection represents a major health threat. Current FDA-approved drugs do not cure HBV. Targeting HBV core protein (Cp) provides an attractive approach toward HBV inhibition and possibly infection cure. We have previously identified and characterized a 5-amino-3-methylthiophene-2,4-dicarboxamide (ATDC) compound as a structurally novel hit for capsid assembly effectors (CAEs). We report herein hit validation through studies on absorption, distribution, metabolism and excretion (ADME) properties and pharmacokinetics (PK), and hit optimization via analogue synthesis aiming to probe the structure-activity relationship (SAR) and structure-property relationship (SPR). In the end, these medicinal chemistry efforts led to the identification of multiple analogues strongly binding to Cp, potently inhibiting HBV replication in nanomolar range without cytotoxicity, and exhibiting good oral bioavailability (F). Two of our analogues, 19o (EC50 = 0.11 µM, CC50 > 100 µM, F = 25%) and 19k (EC50 = 0.31 µM, CC50 > 100 µM, F = 46%), displayed overall lead profiles superior to reported CAEs 7-10 used in our studies.

In Vitro and In Vivo Antimetastatic Effect of Glutathione Disulfide Liposomes.

Cancer metastasis is the major cause of cancer mortality. Despite extensive research efforts, effective treatment for cancer metastasis is still lacking. Cancer metastasis involves 4 essential steps: cell detachment, migration, invasion, and adhesion. Detachment is the first and required step for metastasis. Glutathione disulfide (GSSG) is derived from the oxidation of glutathione (GSH), which is present in biological systems in millimolar concentration. Although GSSG is commercially available, the impact of GSSG on cell functions/dysfunctions has not been fully explored due to the fact that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed GSSG liposomes that effectively deliver GSSG to cells. Unexpectedly, cells treated with GSSG liposomes were resistant to detachment by trypsinization. This observation led to the investigation of the antimetastatic effect of GSSG liposomes. Our data demonstrate that GSSG liposomes at 1 mg/mL completely blocked cell detachment and migration, and significantly inhibited cancer cell invasion. Aqueous GSSG showed no such effect, confirming that the effects on cell detachment, migration, and invasion were caused by the intracellular delivery of GSSG. An in vivo experiment with a murine melanoma experimental metastasis model showed that GSSG liposomes prevented melanoma lung metastasis. The unique antimetastatic mechanism through the effects on detachment and migration, and effective in vitro and in vivo metastasis inhibition, warrants further investigation of the GSSG liposomes as a potential treatment for cancer metastasis.

N-(1-Benzyl-3,5-dimethyl-1H-pyrazol-4-yl)benzamides: Antiproliferative Activity and Effects on mTORC1 and Autophagy.

Guided by antiproliferative activity in MIA PaCa-2 cells, we have performed preliminary structure-activity relationship studies on N-(1-benzyl-3,5-dimethyl-1H-pyrazol-4-yl)benzamides. Two selected compounds showed submicromolar antiproliferative activity and good metabolic stability. Both compounds reduced mTORC1 activity and increased autophagy at the basal level. In addition, they disrupted autophagic flux by interfering with mTORC1 reactivation and clearance of LC3-II under starvation/refeed conditions, as evidenced by accumulation of LC3-II and abnormal LC3 labeled punctae. Therefore, N-(1-benzyl-3,5-dimethyl-1H-pyrazol-4-yl)benzamides may represent a new class of autophagy modulators that possesses potent anticancer activity and potentially a novel mechanism of action.

A fluorescence-based high-throughput assay to identify inhibitors of tyrosylprotein sulfotransferase activity.

Tyrosylprotein sulfotransferases (TPSTs) are Golgi-resident enzymes that catalyze the transfer of a sulfuryl group to the side chain hydroxyl of tyrosine residues. Sulfotyrosine residues are involved in protein-protein interactions in the extracellular space. These interactions are important for chemokines to bind cognate receptor, for cell adhesion and trafficking, and for pathogen entry into cells. To better understand the role of TPSTs in cellular processes and disease states, we are interested in identifying small molecules to modulate TPST activity in experimental systems. Towards that end, we developed a fluorescent peptide assay for TPST2 activity. Here, we demonstrate that this assay can be used to screen the 1280 compound LOPAC library in a 384-well format and in a high-throughput manner. We identified 19 primary hits for a hit rate of 1.5%. Three of the primary hits were verified by dose-response assay and confirmed as inhibitors by a secondary mass spectrometry assay for TPST activity. One hit, suramin, possessed inhibitory properties consistent with a competitive inhibitor of substrate binding and molecular docking revealed a good fit into the TPST2 substrate-binding pocket. This assay can be used to screen larger libraries to identify small molecules that inhibit TPST sulfotransferase activity.

Eeyarestatin I derivatives with improved aqueous solubility.

Inhibition of p97 (also known as valosin-containing protein (VCP)), has been validated as a promising strategy for cancer therapy. Eeyarestatin I (EerI) blocks p97 through a novel mechanism of action and has favorable anti-cancer activities against cultured cancer cells. However, its poor aqueous solubility severely limits its in vivo applications. To circumvent this problem, we have identified EerI derivatives that possess improved aqueous solubility by introducing a single solubilizing group. These modified compounds preserved endoplasmic reticulum (ER) stress-inducing and antiproliferative activities as well as generally good in vitro metabolic properties, suggesting that these EerI derivatives could serve as candidates for further optimization.

Dual anti-HIV mechanism of clofarabine.

BACKGROUND: HIV-1 replication kinetics inherently depends on the availability of cellular dNTPs for viral DNA synthesis. In activated CD4(+) T cells and other rapidly dividing cells, the concentrations of dNTPs are high and HIV-1 reverse transcription occurs in an efficient manner. In contrast, nondividing cells such as macrophages have lower dNTP pools, which restricts efficient reverse transcription. Clofarabine is an FDA approved ribonucleotide reductase inhibitor, which has shown potent antiretroviral activity in transformed cell lines. Here, we explore the potency, toxicity and mechanism of action of clofarabine in the human primary HIV-1 target cells: activated CD4(+) T cells and macrophages. RESULTS: Clofarabine is a potent HIV-1 inhibitor in both activated CD4(+) T cells and macrophages. Due to its minimal toxicity in macrophages, clofarabine displays a selectivity index over 300 in this nondividing cell type. The anti-HIV-1 activity of clofarabine correlated with a significant decrease in both cellular dNTP levels and viral DNA synthesis. Additionally, we observed that clofarabine triphosphate was directly incorporated into DNA by HIV-1 reverse transcriptase and blocked processive DNA synthesis, particularly at the low dNTP levels found in macrophages. CONCLUSIONS: Taken together, these data provide strong mechanistic evidence that clofarabine is a dual action inhibitor of HIV-1 replication that both limits dNTP substrates for viral DNA synthesis and directly inhibits the DNA polymerase activity of HIV-1 reverse transcriptase.

Discovery of 1-hydroxypyridine-2-thiones as selective histone deacetylase inhibitors and their potential application for treating leukemia.

Histone deacetylase (HDAC) is a validated target for pursuing anticancer agents. However, obtaining a selective inhibitor against a given HDAC member remains a significant challenge. We report here the use of 1-hydroxypyridine-2-thione (1HPT) as a key pharmacophore for zinc-binding can result in highly selective HDAC inhibitors. 1HPT-6-carboxylic acid exhibits selective inhibition of HDAC6 with an IC50 of 150 nM that corresponds to a remarkable 0.9 ligand efficiency. Two analogs with simple amino acids shows nearly 600-fold selectivity among the eleven zinc-dependent HDACs. At low micromolar concentration these compounds inhibit the growth of HDAC8-overexpressing chronic myelogenous leukemia cells and specific form of acute myelogenous leukemia cells. Their potential mode of binding was examined by molecular docking and their stability was assessed in mouse and human plasma. Together the results suggest 1HPT analogs exhibit promising therapeutic potential for further development as anticancer agents to treat leukemia.

Evaluation of a dithiocarbamate derivative as a model of thiol oxidative stress in H9c2 rat cardiomyocytes.

Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides and is mainly reflected by the ratio of reduced and oxidized glutathione (GSH/GSSG). A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, as well as thiol modifications such as S-glutathionylation, has been shown to have important implications in a variety of cardiovascular diseases. Therefore, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as examining the impact of pharmacological interventions on thiol pathways. The purpose of this study was to evaluate the use of a dithiocarbamate derivative, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), as a pharmacological model of thiol oxidative stress by examining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including hydrogen peroxide (H2O2), diamide, buthionine sulfoximine, and N,N׳-bis(2-chloroethyl)-N-nitroso-urea. Results indicated that 2-AAPA effectively inhibited glutathione reductase and thioredoxin reductase activities and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as S-glutathionylation. The alteration in TRS led to a loss of mitochondrial membrane potential, release of cytochrome c, and increase in reactive oxygen species production. Compared to other models, 2-AAPA is more potent at creating a state of thiol oxidative stress with lower cytotoxicity, higher specificity, and more pharmacological relevance, and could be utilized as a research tool to study TRS-related normal and abnormal biochemical processes in cardiovascular diseases.