Targeting transcription factors through an IMiD independent zinc finger domain.

Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer.

PROTAC antibiotics: the time is now.

INTRODUCTION: Novel antibiotics are needed to keep antibiotic resistance at bay and to improve treatment of the many drug-susceptible infections for which current therapies achieve poor cure rates. While revolutionizing human therapeutics, the concept of targeted protein degradation (TPD) by bifunctional proteolysis targeting chimeras (PROTACs) has not yet been applied to the discovery of antibiotics. A major obstacle precluding successful translation of this strategy to antibiotic development is that bacteria lack the E3 ligase-proteasome system exploited by human PROTACs to facilitate target degradation. AREAS COVERED: The authors describe the serendipitous discovery of the first monofunctional target-degrading antibiotic pyrazinamide, supporting TPD as a viable and novel approach in antibiotic discovery. They then discuss the rational design, mechanism, and activity of the first bifunctional antibacterial target degrader BacPROTAC, enabling a generalizable approach to TPD in bacteria. EXPERT OPINION: BacPROTACs demonstrate that linking a target directly to a bacterial protease complex can promote target degradation. BacPROTACs successfully bypass the 'middleman' E3 ligase, providing an entry strategy for the generation of antibacterial PROTACs. We speculate that antibacterial PROTACs will not only expand the target space but may also improve treatment by allowing dosage reduction, stronger bactericidal activity and activity against drug-tolerant 'persisters.'

Alkyltriphenylphosphonium turns naphthoquinoneimidazoles into potent membrane depolarizers against mycobacteria.

Due to its central role in energy generation and bacterial viability, mycobacterial bioenergetics is an attractive therapeutic target for anti-tuberculosis drug discovery. Building upon our work on antimycobacterial dioxonaphthoimidazoliums that were activated by a proximal positive charge and generated reactive oxygen species upon reduction by Type II NADH dehydrogenase, we herein studied the effect of a distal positive charge on the antimycobacterial activity of naphthoquinoneimidazoles by incorporating a trialkylphosphonium cation. The potency-enhancing properties of the linker length were affirmed by structure-activity relationship studies. The most active compound against M. tb H37Rv displayed good selectivity index (SI = 34) and strong bactericidal activity in the low micromolar range, which occurred through rapid bacterial membrane depolarization that resulted in depletion of intracellular ATP. Through this work, we demonstrated a switch of the scaffold's mode-of-action via relocation of positive charge while retaining its excellent antibacterial activity and selectivity.

Redox Cycling Dioxonaphthoimidazoliums Disrupt Iron Homeostasis in Mycobacterium bovis Bacillus Calmette-Guérin.

The dioxonaphthoimidazolium scaffold is a novel, highly bactericidal redox cycling antituberculosis chemotype that is reliant on the respiratory enzyme Type II NADH dehydrogenase (NDH2) for the generation of reactive oxygen species (ROS). Here, we employed Mycobacterium bovis Bacillus Calmette-Guérin (M. bovis BCG) reporter strains to show that ROS generated by the redox cycler SA23 simulated an iron deficient state in the bacteria, which led to a compensatory increase in the expression of the iron acquisition mbtB gene while collaterally reducing the expression of the iron storage bfrB gene. Exacerbating the iron deficiency via the inclusion of an iron chelator or aggravating oxidative stress by deploying a catalase (KatG) loss-of-function mutant strain enhanced the activity of SA23, whereas a combined approach of treating the katG mutant strain with an iron chelator led to even greater gains in activity. Our results support the notion that the activity of SA23 pivots on a vicious cycle of events that involve the derailment of iron homeostasis toward greater acquisition of the metal, overwhelmed oxidative stress defenses due to enhanced Fenton reactivity, and, ultimately, self-inflicted death. Hence, we posit that redox cyclers that concurrently perturb the iron equilibrium and cellular respiration are well-positioned to be potent next-generation anti-tubercular drugs. IMPORTANCE Cellular respiration in mycobacteria is a potentially rich target space for the discovery of novel drug entities. Here, we show that a redox cycling bactericidal small molecule that selectively activates a respiratory complex in mycobacteria has the surprising effect of disrupting iron homeostasis. Our results support the notion that the disruption of cellular respiration is a potent driver of reactive oxygen species (ROS) generation by the redox cycling molecule. Mycobacteria respond by acquiring iron to restore the levels depleted by the prevailing oxidizing conditions, which inadvertently trigger the compensatory acquisition of the metal. This leads to overwhelmed oxidative stress defenses and yet more iron depletion. For organisms that are unable to break out of this pernicious cycle of events, cell death is the inevitable outcome. Hence, aberrant ROS production by a redox cycling bactericidal agent inflicts a plethora of damaging effects on mycobacteria, including the derailment of iron homeostasis.

Functionalized Dioxonaphthoimidazoliums: A Redox Cycling Chemotype with Potent Bactericidal Activities against Mycobacterium tuberculosis.

Disruption of redox homeostasis in mycobacteria causes irreversible stress induction and cell death. Here, we report the dioxonaphthoimidazolium scaffold as a novel redox cycling antituberculosis chemotype with potent bactericidal activity against growing and nutrient-starved phenotypically drug-resistant nongrowing bacteria. Maximal potency was dependent on the activation of the redox cycling quinone by the positively charged scaffold and accessibility to the mycobacterial cell membrane as directed by the lipophilicity and conformational characteristics of the N-substituted side chains. Evidence from microbiological, biochemical, and genetic investigations implicates a redox-driven mode of action that is reliant on the reduction of the quinone by type II NADH dehydrogenase (NDH2) for the generation of bactericidal levels of the reactive oxygen species (ROS). The bactericidal profile of a potent water-soluble analogue 32 revealed good activity against nutrient-starved organisms in the Loebel model of dormancy, low spontaneous resistance mutation frequency, and synergy with isoniazid in the checkerboard assay.

High-Content Phenotypic Screen of a Focused TCAMS Drug Library Identifies Novel Disruptors of the Malaria Parasite Calcium Dynamics.

The search for new antimalarial drugs with unexplored mechanisms of action is currently one of the main objectives to combat the resistance already in the clinic. New drugs should target specific mechanisms that once initiated lead inevitably to the parasite's death and clearance and cause minimal toxicity to the host. One such new mode of action recently characterized is to target the parasite's calcium dynamics. Disruption of the calcium homeostasis is associated with compromised digestive vacuole membrane integrity and release of its contents, leading to programmed cell death-like features characterized by loss of mitochondrial membrane potential and DNA degradation. Intriguingly, chloroquine (CQ)-treated parasites were previously reported to exhibit such cellular features. Using a high-throughput phenotypic screen, we identified 158 physiological disruptors (hits) of parasite calcium distribution from a small subset of approximately 3000 compounds selected from the GSK TCAMS (Tres Cantos Anti-Malarial Set) compound library. These compounds were then extensively profiled for biological activity against various CQ- and artemisinin-resistant Plasmodium falciparum strains and stages. The hits were also examined for cytotoxicity, speed of antimalarial activity, and their possible inhibitory effects on heme crystallization. Overall, we identified three compounds, TCMDC-136230, -125431, and -125457, which were potent in inducing calcium redistribution but minimally inhibited heme crystallization. Molecular superimposition of the molecules by computational methods identified a common pharmacophore, with the best fit assigned to TCMDC-125457. There were low cytotoxicity or CQ cross-resistance issues for these three compounds. IC50 values of these three compounds were in the low micromolar range. In addition, TCMDC-125457 demonstrated high efficacy when pulsed in a single-dose combination with artesunate against tightly synchronized artemisinin-resistant ring-stage parasites. These results should add new drug options to the current armament of antimalarial drugs as well as provide promising starting points for development of drugs with non-classical modes of action.

Infigratinib Is a Reversible Inhibitor and Mechanism-Based Inactivator of Cytochrome P450 3A4.

Infigratinib (INF) is a promising selective inhibitor of fibroblast growth factor receptors 1-3 that has recently been accorded both orphan drug designation and priority review status by the US Food and Drug Administration for the treatment of advanced cholangiocarcinoma. Its propensity to undergo bioactivation to electrophilic species was recently expounded upon. However, other than causing aberrant idiosyncratic toxicities, these reactive intermediates may elicit mechanism-based inactivation of cytochrome P450 enzymes. In this study, we investigated the interactions between INF and the most abundant hepatic CYP3A. Our findings revealed that, apart from being a potent noncompetitive reversible inhibitor of CYP3A4, INF inactivated CYP3A4 in a time-, concentration- and NADPH-dependent manner with inactivator concentration at half-maximum inactivation rate constant, maximum inactivation rate constant, and partition ratio of 4.17 µM, 0.068 minute-1, and 41, respectively, when rivaroxaban was employed as the probe substrate. Coincubation with testosterone (alternative CYP3A substrate) or ketoconazole (direct CYP3A inhibitor) attenuated the rate of inactivation, whereas the inclusion of glutathione and catalase did not confer such protection. The lack of enzyme activity recovery after dialysis for 4 hours and oxidation with potassium ferricyanide, coupled with the absence of the characteristic Soret peak signature collectively substantiated that inactivation of CYP3A4 by INF was not mediated by the formation of quasi-irreversible metabolite-intermediate complexes but rather through irreversible covalent adduction to the prosthetic heme and/or apoprotein. Finally, glutathione trapping and high-resolution mass spectrometry experimental results unraveled two plausible bioactivation mechanisms of INF arising from the generation of a p-benzoquinonediimine and epoxide reactive intermediate. SIGNIFICANCE STATEMENT: The potential of INF to cause MBI of CYP3A4 was unknown. This study reports the reversible noncompetitive inhibition and irreversible covalent MBI of CYP3A4 by INF and proposes two potential bioactivation pathways implicating p-benzoquinonediimine and epoxide reactive intermediates, following which a unique covalent docking methodology was harnessed to elucidate the structural and molecular determinants underscoring its inactivation. Findings from this study lay the groundwork for future investigation of clinically relevant drug-drug interactions between INF and concomitant substrates of CYP3A4.

Mechanism-Based Inactivation of Cytochrome P450 3A4 and 3A5 by the Fibroblast Growth Factor Receptor Inhibitor Erdafitinib.

Erdafitinib (ERD) is a first-in-class pan inhibitor of fibroblast growth factor receptor 1-4 that has garnered global regulatory approval for the treatment of advanced or metastatic urothelial carcinoma. Although it has been previously reported that ERD elicits time-dependent inhibition (TDI) of cytochrome P450 (P450) 3A4 (CYP3A4), the exact biochemical nature underpinning this observation remains obfuscated. Moreover, it is also uninterrogated if CYP3A5-its highly homologous counterpart-could be susceptible to such interactions. Mechanism-based inactivation (MBI) of P450 is a unique subset of TDI that hinges on prior bioactivation of the drug to a reactive intermediate and possesses profound clinical and toxicological implications due to its irreversible nature. Here, we investigated and confirmed that ERD inactivated both CYP3A isoforms in a time-, concentration-, and NADPH-dependent manner with KI, kinact, and partition ratio of 4.01 and 10.04 µM, 0.120 and 0.045 min-1, and 32 and 55 for both CYP3A4 and CYP3A5, respectively, when rivaroxaban was employed as the probe substrate. Co-incubation with an alternative substrate or direct inhibitor of CYP3A attenuated the rate of inactivation, whereas the addition of glutathione or catalase did not induce such protection. The lack of enzyme activity recovery following dialysis for 4 h and oxidation with potassium ferricyanide combined with the lack of a Soret peak in spectral scans collectively substantiated that ERD is an irreversible covalent MBI of CYP3A. Finally, glutathione trapping and high-resolution mass spectrometry experiments illuminated a plausible bioactivation mechanism of ERD by CYP3A arising from metabolic epoxidation of its quinoxaline ring.

Amide-Amine Replacement in Indole-2-carboxamides Yields Potent Mycobactericidal Agents with Improved Water Solubility.

Indolecarboxamides are potent but poorly soluble mycobactericidal agents. Here we found that modifying the incipient scaffold by amide-amine substitution and replacing the indole ring with benzothiophene or benzoselenophene led to striking (10-20-fold) improvements in solubility. Potent activity could be achieved without the carboxamide linker but not in the absence of the indole ring. The indolylmethylamine, N-cyclooctyl-6-trifluoromethylindol-2-ylmethylamine (33, MIC90Mtb 0.13 µM, MBC99.9Mtb 0.63 µM), exemplifies a promising member that is more soluble and equipotent to its carboxamide equivalent. It is also an inhibitor of the mycolate transporter MmpL3, a property shared by the methylamines of benzothiophene and benzoselenophene.

Effects of Antimalarial Drugs on Neuroinflammation-Potential Use for Treatment of COVID-19-Related Neurologic Complications.

The SARS-CoV-2 virus that is the cause of coronavirus disease 2019 (COVID-19) affects not only peripheral organs such as the lungs and blood vessels, but also the central nervous system (CNS)-as seen by effects on smell, taste, seizures, stroke, neuropathological findings and possibly, loss of control of respiration resulting in silent hypoxemia. COVID-19 induces an inflammatory response and, in severe cases, a cytokine storm that can damage the CNS. Antimalarials have unique properties that distinguish them from other anti-inflammatory drugs. (A) They are very lipophilic, which enhances their ability to cross the blood-brain barrier (BBB). Hence, they have the potential to act not only in the periphery but also in the CNS, and could be a useful addition to our limited armamentarium against the SARS-CoV-2 virus. (B) They are non-selective inhibitors of phospholipase A2 isoforms, including cytosolic phospholipase A2 (cPLA2). The latter is not only activated by cytokines but itself generates arachidonic acid, which is metabolized by cyclooxygenase (COX) to pro-inflammatory eicosanoids. Free radicals are produced in this process, which can lead to oxidative damage to the CNS. There are at least 4 ways that antimalarials could be useful in combating COVID-19. (1) They inhibit PLA2. (2) They are basic molecules capable of affecting the pH of lysosomes and inhibiting the activity of lysosomal enzymes. (3) They may affect the expression and Fe2+/H+ symporter activity of iron transporters such as divalent metal transporter 1 (DMT1), hence reducing iron accumulation in tissues and iron-catalysed free radical formation. (4) They could affect viral replication. The latter may be related to their effect on inhibition of PLA2 isoforms. Inhibition of cPLA2 impairs an early step of coronavirus replication in cell culture. In addition, a secretory PLA2 (sPLA2) isoform, PLA2G2D, has been shown to be essential for the lethality of SARS-CoV in mice. It is important to take note of what ongoing clinical trials on chloroquine and hydroxychloroquine can eventually tell us about the use of antimalarials and other anti-inflammatory agents, not only for the treatment of COVID-19, but also for neurovascular disorders such as stroke and vascular dementia.

Resistance against Membrane-Inserting MmpL3 Inhibitor through Upregulation of MmpL5 in Mycobacterium tuberculosis.

Spiroketal indolyl Mannich bases (SIMBs) present a novel class of membrane-inserting antimycobacterials with efficacy in a tuberculosis mouse model. SIMBs exert their antibacterial activity by two mechanisms. The indolyl Mannich base scaffold causes permeabilization of bacteria, and the spiroketal moiety contributes to inhibition of the mycolic acid transporter MmpL3. Here, we show that low-level resistance to SIMBs arises by mutations in the transcriptional repressor MmpR5, resulting in upregulation of the efflux pump MmpL5.

Potency Increase of Spiroketal Analogs of Membrane Inserting Indolyl Mannich Base Antimycobacterials Is Due to Acquisition of MmpL3 Inhibition.

Chemistry campaigns identified amphiphilic indolyl Mannich bases as novel membrane-permeabilizing antimycobacterials. Spiroketal analogs of this series showed increased potency, and the lead compound 1 displayed efficacy in a mouse model of tuberculosis. Yet the mechanism by which the spiroketal moiety accomplished the potency "jump" remained unknown. Consistent with its membrane-permeabilizing mechanism, no resistant mutants could be isolated against indolyl Mannich base 2 lacking the spiroketal moiety. In contrast, mutations resistant against spiroketal analog 1 were obtained in mycobacterial membrane protein large 3 (MmpL3), a proton motive force (PMF)-dependent mycolate transporter. Thus, we hypothesized that the potency jump observed for 1 may be due to MmpL3 inhibition acquired by the addition of the spiroketal moiety. Here we showed that 1 inhibited MmpL3 flippase activity without loss of the PMF, colocalized with MmpL3tb-GFP in intact organisms, and yielded a consistent docking pose within the "common inhibitor binding pocket" of MmpL3. The presence of the spiroketal motif in 1 ostensibly augmented its interaction with MmpL3, an outcome not observed in the nonspiroketal analog 2, which displayed no cross-resistance to mmpL3 mutants, dissipated the PMF, and docked poorly in the MmpL3 binding pocket. Surprisingly, 2 inhibited MmpL3 flippase activity, which may be an epiphenomenon arising from its wider membrane disruptive effects. Hence, we conclude that the potency increase associated with the spiroketal analog 1 is linked to the acquisition of a second mechanism, MmpL3 inhibition. In contrast, the nonspiroketal analog 2 acts pleiotropically, affecting several cell membrane-embedded targets, including MmpL3, through its membrane permeabilizing and depolarizing effects.

Extreme Drug Tolerance of Mycobacterium abscessus "Persisters".

Persistence of infection despite extensive chemotherapy with antibiotics displaying low MICs is a hallmark of lung disease caused by Mycobacterium abscessus (Mab). Thus, the classical MIC assay is a poor predictor of clinical outcome. Discovery of more efficacious antibiotics requires more predictive in vitro potency assays. As a mycobacterium, Mab is an obligate aerobe and a chemo-organo-heterotroph - it requires oxygen and organic carbon sources for growth. However, bacteria growing in patients can encounter micro-environmental conditions that are different from aerated nutrient-rich broth used to grow planktonic cultures for MIC assays. These in vivo conditions may include oxygen and nutrient limitation which should arrest growth. Furthermore, Mab was shown to grow as biofilms in vivo. Here, we show Mab Bamboo, a clinical isolate we use for Mab drug discovery, can survive oxygen deprivation and nutrient starvation for extended periods of time in non-replicating states and developed an in vitro model where the bacterium grows as biofilm. Using these culture models, we show that non-replicating or biofilm-growing bacteria display tolerance to clinically used anti-Mab antibiotics, consistent with the observed persistence of infection in patients. To demonstrate the utility of the developed "persister" assays for drug discovery, we determined the effect of novel agents targeting membrane functions against these physiological forms of the bacterium and find that these compounds show "anti-persister" activity. In conclusion, we developed in vitro "persister" assays to fill an assay gap in Mab drug discovery compound progression and to enable identification of novel lead compounds showing "anti-persister" activity.

Rifabutin Suppresses Inducible Clarithromycin Resistance in Mycobacterium abscessus by Blocking Induction of whiB7 and erm41.

Clarithromycin (CLR) is the corner stone in regimens for the treatment of lung disease caused by Mycobacterium abscessus (Mab). However, many strains harbor the CLR-inducible CLR resistance gene erm41, encoding a ribosome methylase. Induction of erm41 is mediated by the transcription factor whiB7. We hypothesized that an inhibitor of RNA synthesis should be able to block the whiB7-erm41 induction response to CLR exposure and thus suppress CLR resistance. Recently, we discovered that the rifampicin analog rifabutin (RFB) shows attractive potency against Mab. To determine whether RFB-CLR combinations are synergistic, a checkerboard analysis against a collection of erm41 positive and negative Mab strains was carried out. This revealed synergy of the two drugs for erm41 positive but not for erm41 negative strains. To determine whether RFB's potentiation effect was due to inhibition of the transcriptional induction of the whiB7-erm41 resistance system, we measured the effect of CLR alone and in combination with RFB on whiB7 and erm41 mRNA levels. CLR alone strongly induced whiB7 and erm41 expression as expected. The synergistic, growth-inhibiting combination of RFB with CLR blocked induction of both genes. These results suggest that RFB suppresses inducible CLR resistance by preventing induction of whiB7 and erm41 expression.

Antimalarial N 1,N 3-Dialkyldioxonaphthoimidazoliums: Synthesis, Biological Activity, and Structure-activity Relationships.

Here we report the nanomolar potencies of N 1,N 3-dialkyldioxonaphthoimidazoliums against asexual forms of sensitive and resistant Plasmodium falciparum. Activity was dependent on the presence of the fused quinone-imidazolium entity and lipophilicity imparted by the N1/N3 alkyl residues on the scaffold. Gametocytocidal activity was also detected, with most members active at IC50 < 1 µM. A representative analog with good solubility, limited PAMPA permeability, and microsomal stability demonstrated oral efficacy on a humanized mouse model of P. falciparum.

Galloyl esters of trans-stilbenes are inhibitors of FASN with anticancer activity on non-small cell lung cancer cells.

Fatty acid synthase (FASN) is a lipogenic enzyme that is selectively upregulated in malignant cells. There is growing consensus on the oncogenicity of FASN-driven lipogenesis and the potential of FASN as a druggable target in cancer. Here, we report the synthesis and FASN inhibitory activities of two novel galloyl esters of trans-stilbene EC1 and EC5. Inhibition of FASN was accompanied by a loss in AKT activation and profound apoptosis in several non-small cell lung cancer (NSCLC) cells at the growth inhibitory concentrations of EC1 and EC5. Both FASN and phospho-AKT levels were concurrently downregulated. However, addition of a lipid concentrate to the treated cells reinstated cell viability and reversed the loss of FASN and AKT protein levels, thus recapitulating the causal relationship between FASN inhibition and the loss in cell viability.

Gut Microbiota Metabolite Indole Propionic Acid Targets Tryptophan Biosynthesis in Mycobacterium tuberculosis.

Indole propionic acid (IPA), produced by the gut microbiota, is active against Mycobacterium tuberculosisin vitro and in vivo However, its mechanism of action is unknown. IPA is the deamination product of tryptophan (Trp) and thus a close structural analog of this essential aromatic amino acid. De novo Trp biosynthesis in M. tuberculosis is regulated through feedback inhibition: Trp acts as an allosteric inhibitor of anthranilate synthase TrpE, which catalyzes the first committed step in the Trp biosynthesis pathway. Hence, we hypothesized that IPA may mimic Trp as an allosteric inhibitor of TrpE and exert its antimicrobial effect by blocking synthesis of Trp at the TrpE catalytic step. To test our hypothesis, we carried out metabolic, chemical rescue, genetic, and biochemical analyses. Treatment of mycobacteria with IPA inhibited growth and reduced the intracellular level of Trp, an effect abrogated upon supplementation of Trp in the medium. Missense mutations at the allosteric Trp binding site of TrpE eliminated Trp inhibition and caused IPA resistance. In conclusion, we have shown that IPA blocks Trp biosynthesis in M. tuberculosis via inhibition of TrpE by mimicking the physiological allosteric inhibitor of this enzyme.IMPORTANCE New drugs against tuberculosis are urgently needed. The tryptophan (Trp) analog indole propionic acid (IPA) is the first antitubercular metabolite produced by human gut bacteria. Here, we show that this antibiotic blocks Trp synthesis, an in vivo essential biosynthetic pathway in M. tuberculosis Intriguingly, IPA acts by decoupling a bacterial feedback regulatory mechanism: it mimics Trp as allosteric inhibitor of anthranilate synthase, thereby switching off Trp synthesis regardless of intracellular Trp levels. The identification of IPA's target paves the way for the discovery of more potent TrpE ligands employing rational, target-based lead optimization.

Anti-survivin effect of the small molecule inhibitor YM155 in RCC cells is mediated by time-dependent inhibition of the NF-κB pathway.

Constitutive activation of the NF-κB signaling cascade is associated with tumourigenesis and poor prognosis in many human cancers including RCC. YM155, a small molecule inhibitor of survivin, was previously shown to potently inhibit the viability of immortalized and patient derived renal cell carcinoma (RCC) cell lines. Here we investigated the role of NF-κB signaling to the anti-cancer properties of YM155 in RCC786.0 cells. YM155 diminished nuclear levels of p65 and phosphorylated p65 and attenuated the transcriptional competencies of the p65/p50 heterodimers. Accordingly, we found that YM155 diminished the transcription of NF-κB target gene survivin. Events that led to the interception of the nuclear translocation of p65/p50 were the activation of the deubiquinating enzyme CYLD by YM155, which led to the inhibition of IKKß, stabilization of IκBα and retention of NF-κB heterodimers in the cytosol. Importantly, the suppressive effects of YM155 were time-dependent and observed at the 24 h time point, and not earlier. TNF-α, a stimulator of NF-κB signaling did not affect its inhibitory properties. The ability of YM155 to intercept a major transcriptional pathway like NF-κB, would have important ramifications on the pharmacodynamics effects elicited by this unusual molecule.

Indolyl Azaspiroketal Mannich Bases Are Potent Antimycobacterial Agents with Selective Membrane Permeabilizing Effects and in Vivo Activity.

The inclusion of an azaspiroketal Mannich base in the membrane targeting antitubercular 6-methoxy-1- n-octyl-1 H-indole scaffold resulted in analogs with improved selectivity and submicromolar activity against Mycobacterium tuberculosis H37Rv. The potency enhancing properties of the spiro-fused ring motif was affirmed by SAR and validated in a mouse model of tuberculosis. As expected for membrane inserting agents, the indolyl azaspiroketal Mannich bases perturbed phospholipid vesicles, permeabilized bacterial cells, and induced the mycobacterial cell envelope stress reporter promoter p iniBAC. Surprisingly, their membrane disruptive effects did not appear to be associated with bacterial membrane depolarization. This profile was not uniquely associated with azaspiroketal Mannich bases but was characteristic of indolyl Mannich bases as a class. Whereas resistant mycobacteria could not be isolated for a less potent indolyl Mannich base, the more potent azaspiroketal analog displayed low spontaneous resistance mutation frequency of 10-8/CFU. This may indicate involvement of an additional envelope-related target in its mechanism of action.

The mechanistic effects of the dioxonaphthoimidazolium analog YM155 in renal cell carcinoma cell cycling and apoptosis.

AIM: To investigate the effect of dioxonaphthoimidazolium analog YM155 on cell cycle progression of the clear-cell variant of renal cell carcinoma (ccRCC). MAIN METHODS: Cell cycle analysis was performed using bromodeoxyuridine (BrdU) and PI, apoptosis initiation was monitored using Annexin V and proteins expression was determined using western immunoblotting. KEY FINDINGS: Here, we showed that YM155 activated stress-related molecules (histone H2AX, checkpoint kinases Chk1 and Chk2, p53) that mediate DNA damage checkpoint responses. The coordinated activation of these effector molecules disrupts progression of the cell cycle at the S phase as deduced from BrdU pulsing experiments and the ensuing changes in the levels of proteins (cyclins, CDKs, CDK inhibitors, phosphatases) that control cell cycle progression. Notably, we found increases in cyclin E and Cdc2 which regulate transition of cells from G1 to S, even as losses were observed for other CDKs and their cyclin partners. Furthermore, by inducing a loss in total pRb possibly by promoting its degradation, YM155 promoted the E2F transcription of genes that regulate entry into the S phase. After 24 h, cell cycle arrest to repair YM155-inflicted DNA damage was overtaken by p53-mediated apoptosis. YM155 induced increases in pro-apoptotic proteins (Bax and Bad), diminished anti-apoptotic proteins (Mcl-1, Bcl-xl, XIAP, survivin) and initiated cleavage of apoptotic marker proteins caspase 3 and PARP. SIGNIFICANCE: Taken together, the added insight provided on the cell cycle perturbative effects of YM155 may assist clinicians in framing rational choices for combining YM155 with other anti-cancer drugs or treatment modalities in ccRCC.