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.

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.

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.

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.

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.

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.

Indolylalkyltriphenylphosphonium Analogues Are Membrane-Depolarizing Mycobactericidal Agents.

Agents that selectively target the mycobacterial membrane could potentially shorten treatment time for tuberculosis, reduce relapse, and curtail emergence of resistant strains. The lipophilicity and extensive charge-delocalized state of the triphenylphosphonium cation strongly favor accumulation within bacterial membranes. Here, we explored the antimycobacterial activities and membrane-targeting properties of indolylalkyltriphenylphosphonium analogues. The most active analogues preferentially inhibited growth of Mycobacterium tuberculosis H37Rv (MIC50 2-4 µM) and were bactericidal against Mycobacterium bovis BCG (MBC99 3 µM). In spite of their propensity to accumulate within membranes, we found no evidence that these compounds permeabilized mycobacterial membranes or induced cell-envelope stress. Our investigations indicated that their bacterical effects stem from sustained depolarization of mycobacterial membranes and ensuing disruptive effects on electron transfer and cell division.

Amphiphilic Indole Derivatives as Antimycobacterial Agents: Structure-Activity Relationships and Membrane Targeting Properties.

Antibacterials that disrupt cell membrane function have the potential to eradicate "persister" organisms and delay the emergence of resistance. Here we report the antimycobacterial activities of 4-fluoro and 6-methoxyindoles bearing a cationic amphiphilic motif represented by a lipophilic n-octyl side chain at position 1 and a positively charged azepanyl or 1,4-dioxa-8-azaspiro[4.5]decane moiety at position 3. These analogues exhibited balanced profiles of potency (Mycobacterium bovis BCG, M tuberculosis H37Rv), selective activity, solubility, and metabolic stability. Bacteriological mechanism of action investigations on a representative analogue revealed cell membrane permeabilization and depolarization in M bovis BCG. These membrane-related changes preceded cell death indicating that the loss in membrane integrity was not an epiphenomenon. Bactericidal activity was observed against both growing and nongrowing mycobacterial cultures. The analogue also upregulated cell envelope stress-inducible promoters piniBAC and pclgR, implicating the involvement of envelope-related targets in its mode of action.

Dioxonaphthoimidazoliums are Potent and Selective Rogue Stem Cell Clearing Agents with SOX2-Suppressing Properties.

Pluripotent stem cells are uniquely positioned for regenerative medicine, but their clinical potential can only be realized if their tumorigenic tendencies are decoupled from their pluripotent properties. Deploying small molecules to remove remnant undifferentiated pluripotent cells, which would otherwise transform into teratomas and teratomacarcinomas, offers several advantages over non-pharmacological methods. Dioxonapthoimidazolium YM155, a survivin suppressant, induced selective and potent cell death of undifferentiated stem cells. Herein, the structural requirements for stemotoxicity were investigated and found to be closely aligned with those essential for cytotoxicity in malignant cells. There was a critical reliance on the quinone and imidazolium moieties but a lesser dependence on ring substituents, which served mainly to fine-tune activity. Several potent analogues were identified which, like YM155, suppressed survivin and decreased SOX2 in stem cells. The decrease in SOX2 would cause an imbalance in pluripotent factors that could potentially prompt cells to differentiate and hence decrease the risk of aberrant teratoma formation. As phosphorylation of the NF-κB p50 subunit was also suppressed, the crosstalk between phospho-p50, SOX2, and survivin could implicate a causal role for NF-κB signaling in mediating the stem cell clearing properties of dioxonaphthoimidazoliums.

Antiproliferative, DNA intercalation and redox cycling activities of dioxonaphtho[2,3-d]imidazolium analogs of YM155: A structure-activity relationship study.

The anticancer agent YM155 is widely investigated as a specific survivin suppressant. More recently, YM155 was found to induce DNA damage and this has raised doubts as to whether survivin is its primary target. In an effort to assess the contribution of DNA damage to the anticancer activity of YM155, several analogs were prepared and evaluated for antiproliferative activity on malignant cells, participation in DNA intercalation and free radical generation by redox cycling. The intact positively charged scaffold was found to be essential for antiproliferative activity and intercalation but was less critical for redox cycling where the minimal requirement was a pared down bicyclic quinone. Side chain requirements at the N(1) and N(3) positions of the scaffold were more alike for redox cycling and intercalation than antiproliferative activity, underscoring yet again, the limited structural overlaps for these activities. Furthermore, antiproliferative activities were poorly correlated to DNA intercalation and redox cycling. Potent antiproliferative activity (IC50 9-23 nM), exceeding that of YM155, was found for a minimally substituted methyl analog AB7. Like YM155 and other dioxonaphthoimidazoliums, AB7 was a modest DNA intercalator but with weak redox cycling activity. Thus, the capacity of this scaffold to inflict direct DNA damage leading to cell death may not be significant and YM155 should not be routinely classified as a DNA damaging agent.

N'-Alkylaminosulfonyl Analogues of 6-Fluorobenzylideneindolinones with Desirable Physicochemical Profiles and Potent Growth Inhibitory Activities on Hepatocellular Carcinoma.

The benzylideneindolinone 6-chloro-3-(3'-trifluoromethylbenzylidene)-1,3-dihydroindol-2-one (4) was reported to exhibit potent and selective growth inhibitory effects on hepatocellular carcinoma (HCC). Corroborative evidence supported multi-receptor tyrosine kinase (RTK) inhibition as a possible mode of action. However, the poor physicochemical properties of 4 limited its furtherance as a lead compound. In this study, the modification of 4 was investigated with the aim of improving its potency and physicochemical profile. The 6-fluorobenzylideneindolinone 3-12 bearing a 3'-N-propylaminosulfonyl substituent was found to be a promising substitute. Compound 3-12 [6-fluoro-3-(3'-N-propylaminosulfonylbenzylidene)-1,3-dihydroindol-2-one] was found to be tenfold more soluble than 4 and to have sub-micromolar growth inhibitory activities on HCC cells. It is apoptogenic and inhibits the phosphorylation of several RTKs in HuH7, of which the inhibition of FGFR4 and HER3 are prominent. Compound 3-12 decreased the tumor load in a physiologically relevant orthotopic HCC xenograft murine model. Structure-activity relationships support pivotal roles for the fluoro and N'-propylaminosulfonyl moieties in enhancing cell-based activity and moderating the physicochemical profile (solubility, permeability) of 3-12.

Determining the Functions of HIV-1 Tat and a Second Magnesium Ion in the CDK9/Cyclin T1 Complex: A Molecular Dynamics Simulation Study.

The current paradigm of cyclin-dependent kinase (CDK) regulation based on the well-established CDK2 has been recently expanded. The determination of CDK9 crystal structures suggests the requirement of an additional regulatory protein, such as human immunodeficiency virus type 1 (HIV-1) Tat, to exert its physiological functions. In most kinases, the exact number and roles of the cofactor metal ions remain unappreciated, and the repertoire has thus gained increasing attention recently. Here, molecular dynamics (MD) simulations were implemented on CDK9 to explore the functional roles of HIV-1 Tat and the second Mg2+ ion at site 1 (Mg12+). The simulations unveiled that binding of HIV-1 Tat to CDK9 not only stabilized hydrogen bonds (H-bonds) between ATP and hinge residues Asp104 and Cys106, as well as between ATP and invariant Lys48, but also facilitated the salt bridge network pertaining to the phosphorylated Thr186 at the activation loop. By contrast, these H-bonds cannot be formed in CDK9 owing to the absence of HIV-1 Tat. MD simulations further revealed that the Mg12+ ion, coupled with the Mg22+ ion, anchored to the triphosphate moiety of ATP in its catalytic competent conformation. This observation indicates the requirement of the Mg12+ ion for CDK9 to realize its function. Overall, the introduction of HIV-1 Tat and Mg12+ ion resulted in the active site architectural characteristics of phosphorylated CDK9. These data highlighted the functional roles of HIV-1 Tat and Mg12+ ion in the regulation of CDK9 activity, which contributes an important complementary understanding of CDK molecular underpinnings.

Functionalized tetrahydro-1H-pyrido[4,3-b]indoles: a novel chemotype with Sirtuin 2 inhibitory activity.

Sirtuins are protein deacylases with regulatory roles in metabolism and stress response. Functionalized tetrahydro-1H-pyrido[4,3-b]indoles were identified as preferential sirtuin 2 inhibitors, with in vitro inhibitory potencies in the low micromolar concentrations (IC50 3-4 µM) for the more promising candidates. The functional relevance of sirtuin inhibition was corroborated in western blots that showed hyperacetylation of p53 and α-tubulin in treated HepG2 and MDA-MB-231 cells. Molecular docking showed that the tetrahydropyridoindole scaffold was positioned in the NAD + pocket and the acetylated substrate channel of the sirtuin 2 protein by van der Waals/hydrophobic, H bonding and stacking interactions. Functionalized tetrahydropyridoindoles represent a novel class of sirtuin 2 inhibitors that could be further explored for its therapeutic potential.

An improved isoprenylcysteine carboxylmethyltransferase inhibitor induces cancer cell death and attenuates tumor growth in vivo.

Inhibitors of isoprenylcysteine carboxylmethyltransferase (Icmt) are promising anti-cancer agents, as modification by Icmt is an essential component of the protein prenylation pathway for a group of proteins that includes Ras GTPases. Cysmethynil, a prototypical indole-based inhibitor of Icmt, effectively inhibits tumor cell growth. However, the physical properties of cysmethynil, such as its low aqueous solubility, make it a poor candidate for clinical development. A novel amino-derivative of cysmethynil with superior physical properties and marked improvement in efficacy, termed compound 8.12, has recently been reported. We report here that Icmt (-/-) mouse embryonic fibroblasts (MEFs) are much more resistant to compound 8.12-induced cell death than their wild-type counterparts, providing evidence that the anti-proliferative effects of this compound are mediated through an Icmt specific mechanism. Treatment of PC3 prostate and HepG2 liver cancer cells with compound 8.12 resulted in pre-lamin A accumulation and Ras delocalization from the plasma membrane, both expected outcomes from inhibition of the Icmt-catalyzed carboxylmethylation. Treatment with compound 8.12 induced cell cycle arrest, autophagy and cell death, and abolished anchorage-independent colony formation. Consistent with its greater in vitro efficacy, compound 8.12 inhibited tumor growth with greater potency than cysmethynil in a xenograft mouse model. Further, a drug combination study identified synergistic antitumor efficacy of compound 8.12 and the epithelial growth factor receptor (EGFR)-inhibitor gefitinib, possibly through enhancement of autophagy. This study establishes compound 8.12 as a pharmacological inhibitor of Icmt that is an attractive candidate for further preclinical and clinical development.

Synthesis and evaluation of bisbenzylidenedioxotetrahydrothiopranones as activators of endoplasmic reticulum (ER) stress signaling pathways and apoptotic cell death in acute promyelocytic leukemic cells.

Curcumin is known to trigger ER-stress induced cell death of acute promyelocytic leukemic (APL) cells by intercepting the degradation of nuclear co-repressor (N-CoR) protein which has a key role in the pathogenesis of APL. Replacing the heptadienedione moiety of curcumin with a monocarbonyl cross-conjugated dienone embedded in a tetrahydrothiopyranone dioxide ring resulted in thiopyranone dioxides that were more resilient to hydrolysis and had greater growth inhibitory activities than curcumin on APL cells. Several members intercepted the degradation of misfolded N-CoR and triggered the signaling cascade in the unfolded protein response (UPR) which led to apoptotic cell death. Microarray analysis showed that genes involved in protein processing pathways that were germane to the activation of the UPR were preferentially up-regulated in treated APL cells, supporting the notion that the UPR was a consequential mechanistic pathway affected by thiopyranone dioxides. The Michael acceptor reactivity of the scaffold may have a role in exacerbating ER stress in APL cells.

Functionalized acridin-9-yl phenylamines protected neuronal HT22 cells from glutamate-induced cell death by reducing intracellular levels of free radical species.

The in vitro neuronal cell death model based on the HT22 mouse hippocampal cell model is a convenient means of identifying compounds that protect against oxidative glutamate toxicity which plays a role in the development of certain neurodegenerative diseases. Functionalized acridin-9-yl-phenylamines were found to protect HT22 cells from glutamate challenge at submicromolar concentrations. The Aryl(1)-NH-Aryl(2) scaffold that is embedded in these compounds was the minimal pharmacophore for activity. Mechanistically, protection against the endogenous oxidative stress generated by glutamate did not involve up-regulation of glutathione levels but attenuation of the late stage increases in mitochondrial ROS and intracellular calcium levels. The NH residue in the pharmacophore played a crucial role in this regard as seen from the loss of neuroprotection when it was structurally modified or replaced. That the same NH was essential for radical scavenging in cell-free and cell-based systems pointed to an antioxidant basis for the neuroprotective activities of these compounds.