Plitidepsin as an Immunomodulator against Respiratory Viral Infections.

Plitidepsin is a host-targeted compound known for inducing a strong anti-SARS-CoV-2 activity, as well as for having the capacity of reducing lung inflammation. Because IL-6 is one of the main cytokines involved in acute respiratory distress syndrome, the effect of plitidepsin in IL-6 secretion in different in vitro and in vivo experimental models was studied. A strong plitidepsin-mediated reduction of IL-6 was found in human monocyte-derived macrophages exposed to nonproductive SARS-CoV-2. In resiquimod (a ligand of TLR7/8)-stimulated THP1 human monocytes, plitidepsin-mediated reductions of IL-6 mRNA and IL-6 levels were also noticed. Additionally, although resiquimod-induced binding to DNA of NF-κB family members was unaffected by plitidepsin, a decrease in the regulated transcription by NF-κB (a key transcription factor involved in the inflammatory cascade) was observed. Furthermore, the phosphorylation of p65 that is required for full transcriptional NF-κB activity was significantly reduced by plitidepsin. Moreover, decreases of IL-6 levels and other proinflammatory cytokines were also seen in either SARS-CoV-2 or H1N1 influenza virus-infected mice, which were treated at low enough plitidepsin doses to not induce antiviral effects. In summary, plitidepsin is a promising therapeutic agent for the treatment of viral infections, not only because of its host-targeted antiviral effect, but also for its immunomodulatory effect, both of which were evidenced in vitro and in vivo by the decrease of proinflammatory cytokines.

Enigmazole C: A Cytotoxic Macrocyclic Lactone and Its Ring-Opened Derivatives from a New Species of Homophymia Sponge.

A new macrolide, enigmazole C (1), and two additional analogues, enigmazoles E (2) and D (3), were obtained from a new species of the Homophymia genus as part of an ongoing discovery program at PharmaMar to study cytotoxic substances from marine sources. The structures were fully characterized by cumulative analyses of NMR, IR, and MS spectra, along with density functional theory computational calculations. All three of the new compounds feature an unusual 2,3-dihydro-4H-pyran-4-one moiety, but only enigmazoles C (1) and D (3) showed cytotoxic activity in the micromolar range against A-549 (lung), HT-29 (colon), MDA-MB-231 (breast), and PSN-1 (pancreas) tumor cells.

Identification of trans-AT polyketide clusters in two marine bacteria reveals cryptic similarities between distinct symbiosis factors.

Glutarimide-containing polyketides are known as potent antitumoral and antimetastatic agents. The associated gene clusters have only been identified in a few Streptomyces producers and Burkholderia gladioli symbiont. The new glutarimide-family polyketides, denominated sesbanimides D, E and F along with the previously known sesbanimide A and C, were isolated from two marine alphaproteobacteria Stappia indica PHM037 and Labrenzia aggregata PHM038. Structures of the isolated compounds were elucidated based on 1D and 2D homo and heteronuclear NMR analyses and ESI-MS spectrometry. All compounds exhibited strong antitumor activity in lung, breast and colorectal cancer cell lines. Subsequent whole genome sequencing and genome mining revealed the presence of the trans-AT PKS gene cluster responsible for the sesbanimide biosynthesis, described as sbn cluster. Strikingly, the modular architecture of downstream mixed type PKS/NRPS, SbnQ, revealed high similarity to PedH in pederin and Lab13 in labrenzin gene clusters, although those clusters are responsible for the production of structurally completely different molecules. The unexpected presence of SbnQ homologues in unrelated polyketide gene clusters across phylogenetically distant bacteria, raises intriguing questions about the evolutionary relationship between glutarimide-like and pederin-like pathways, as well as the functionality of their synthetic products.

Unique Polyhalogenated Peptides from the Marine Sponge Ircinia sp.

Two new bromopyrrole peptides, haloirciniamide A (1) and seribunamide A (2), have been isolated from an Indonesian marine sponge of the genus Ircinia collected in the Thousand Islands (Indonesia). The planar structure of both compounds was assigned on the basis of extensive 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configuration of the amino acid residues in 1 and 2 was determined by the application of Marfey's method. Compound 1 is the first dibromopyrrole cyclopeptide having a chlorohistidine ring, while compound 2 is a rare peptide possessing a tribromopyrrole ring. Both compounds failed to show significant cytotoxicity against four human tumor cell lines, and neither compound was able to inhibit the enzyme topoisomerase I or impair the interaction between programmed cell death protein PD1 and its ligand, PDL1.

Bioconjugation through Mesitylene Thiol Alkylation.

The design and generation of complex multifunctional macromolecular structures by bioconjugation is a hot topic due to increasing interest in conjugates with therapeutic applications. In this regard, the development of efficient, selective, and safe conjugation methods is a major objective. In this report, we describe the use of the bis(bromomethyl)benzene scaffold as a linker for bioconjugation with special emphasis on antibody conjugation. We first performed the monothioalkylation of 1,3,5-tris(bromomethyl)benzene, which rendered the reactive dibromotrimethylbenzyl derivatives to be used in thiol bis-alkylation. Next, we introduced into the linker either a bis(Cys)-containing peptide or anti-CD4 and -CD13 monoclonal antibodies, previously subjected to partial reduction of disulfide bonds. Mass spectrometry, UV-vis spectra, and SDS-PAGE experiments revealed that this bis-alkylating agent for bioconjugation preserved both antibody integrity and antibody-antigen binding affinity, as assessed by flow cytometry. Taken together, our results show that the mesitylene scaffold is a suitable linker for thiol-based bioconjugation reactions. This linker could be applicable in the near future for the preparation of antibody drug conjugates.

Can Stereoclusters Separated by Two Methylene Groups Be Related by DFT Studies? The Case of the Cytotoxic Meroditerpenes Halioxepines.

QM/NMR-DFT (quantum mechanics combined with nuclear magnetic resonance parameters calculated by density functional theory approximations) studies allowed us to link two stereoclusters separated by two methylene groups present in the new meroditerpenes halioxepine B (2) and halioxepine C (3) and the known halioxepine (1), isolated from two Indonesian sponges of the genus Haliclona (Reniera). DP4 and DP4+ probabilities were used to discriminate the two diastereotopic arrangements of the two stereoclusters, whose unconnected relative configurations were determined by ROESY and J-based configurational analysis. To confirm the DFT studies, the full relative configuration of 1 was deduced using a mixture of benzene-d6 and pyridine-d5 as the NMR solvent. ROESY measurements connected the two stereoclusters and demonstrated that DFT calculations accurately predict the configuration when two methylenes separate the two stereoclusters. The different arrangements of the distant stereoclusters C-1/C-2/C-7 and C-10/C-15 for compounds 2 and 3 were deduced by DFT calculations and explained the opposite optical rotations observed for the two compounds. Halioxepines B (2) and C (3) display moderate cytotoxicity against different human cancer cell lines.

Protoxenicins A and B, Cytotoxic Long-Chain Acylated Xenicanes from the Soft Coral Protodendron repens.

Two new xenicanes, named protoxenicins A (1) and B (2), were isolated from an organic extract of the soft coral Protodendron repens, collected off the coast of Okuza (Tanzania), being the first chemical study of an organism belonging to this genus. Their planar structures were determined by 1D and 2D NMR and HRESIMS techniques, while the relative configurations were elucidated by comparison of their chemical shifts and coupling constants with the literature values of their congeners, as well as by ROESY experiments, chemical derivatization, and molecular mechanics calculations. This is the first report of a xenicin acylated with a long saturated fatty acid. Furthermore, the absolute configuration of the stereogenic centers of the cyclononane ring and at C-1 in 1 was determined by Mosher's method. Protoxenicin B (2) is present in solution as a mixture of two conformers in a 2:1 ratio deduced by 1H NMR. Both xenicanes display significant cytotoxic activity against a panel of different tumor cell lines.

Streptenols F-I Isolated from the Marine-Derived Streptomyces misionensis BAT-10-03-023.

A marine-derived bacterium, Streptomyces misionensis BAT-10-03-123, has produced four new streptenol derivatives, F, G, H, and I (1-4), as well as the known streptenols A and C (5 and 6). Their planar structures were elucidated by detailed analysis of spectroscopic data. The absolute configurations of the new streptenol compounds were determined by chemical and spectroscopic methods, including Mosher's ester method. All of the compounds were tested for cytotoxicity against four selected cancer cell lines.

Daedophamide, a Cytotoxic Cyclodepsipeptide from a Daedalopelta sp. Sponge Collected in Indonesia.

A new cyclodepsipeptide, daedophamide (1), has been isolated from a Daedalopelta sp. marine sponge collected from Alor Island (Indonesia). The planar structure of 1 was assigned on the basis of extensive 1D and 2D NMR spectroscopy and mass spectrometry. Daedophamide (1) contains 11 amino acid residues and an amide-linked 3-hydroxy-2,4,6,8-tetramethylnonanoic acid (Htemna). The amino acid constituents were identified as l-Leu, N-Me-l-Gln, d-Arg, d-Asp, d-allo-Thr, l-Pip, d-Ala, d-Ser, 3,4-dimethyl-Gln, O-MeThr, and 4-amino-7-guanidino-2,3-dihydroxyheptanoic acid (Agdha). The absolute configurations of eight of the amino acid residues in 1 were determined by application of the Marfey's method after acid-catalyzed hydrolysis, with the relative configurations of the remaining three amino acid residues and the Htemna unit being assigned by comparison of the NMR data with those reported for other similar peptides. Compound 1 displayed strong cytotoxic activity against a panel of four human tumor cell lines with GI50 values in the submicromolar range.

Bacterial Production of a Pederin Analogue by a Free-Living Marine Alphaproteobacterium.

The polyketide pederin family are cytotoxic compounds isolated from insects, lichen, and marine sponges. During the past decade, different uncultivable bacteria symbionts have been proposed as the real producers of these compounds, such as those found in insects, lichen, and marine sponges, and their trans-AT polyketide synthase gene clusters have been identified. Herein we report the isolation and biological activities of a new analogue of the pederin family, compound 1, from the culture of a marine heterotrophic alphaproteobacterium, Labrenzia sp. PHM005. This is the first report of the production of a pederin-type compound by a free-living marine bacteria that could be cultured in the laboratory.

Bistratamides M and N, Oxazole-Thiazole Containing Cyclic Hexapeptides Isolated from Lissoclinum bistratum Interaction of Zinc (II) with Bistratamide K.

Two novel oxazole-thiazole containing cyclic hexapeptides, bistratamides M (1) and N (2) have been isolated from the marine ascidian Lissoclinum bistratum (L. bistratum) collected in Raja Ampat (Papua Bar, Indonesia). The planar structure of 1 and 2 was assigned on the basis of extensive 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configuration of the amino acid residues in 1 and 2 was determined by the application of the Marfey's and advanced Marfey's methods after ozonolysis followed by acid-catalyzed hydrolysis. The interaction between zinc (II) and the naturally known bistratamide K (3), a cyclic hexapeptide isolated from a different specimen of Lissoclinum bistratum, was monitored by ¹H and 13C NMR. The results obtained are consistent with the proposal that these peptides are biosynthesized for binding to metal ions. Compounds 1 and 2 display moderate cytotoxicity against four human tumor cell lines with GI50 values in the micromolar range.

A new tubulin-binding site and pharmacophore for microtubule-destabilizing anticancer drugs.

The recent success of antibody-drug conjugates (ADCs) in the treatment of cancer has led to a revived interest in microtubule-destabilizing agents. Here, we determined the high-resolution crystal structure of the complex between tubulin and maytansine, which is part of an ADC that is approved by the US Food and Drug Administration (FDA) for the treatment of advanced breast cancer. We found that the drug binds to a site on ß-tubulin that is distinct from the vinca domain and that blocks the formation of longitudinal tubulin interactions in microtubules. We also solved crystal structures of tubulin in complex with both a variant of rhizoxin and the phase 1 drug PM060184. Consistent with biochemical and mutagenesis data, we found that the two compounds bound to the same site as maytansine and that the structures revealed a common pharmacophore for the three ligands. Our results delineate a distinct molecular mechanism of action for the inhibition of microtubule assembly by clinically relevant agents. They further provide a structural basis for the rational design of potent microtubule-destabilizing agents, thus opening opportunities for the development of next-generation ADCs for the treatment of cancer.

Insulin resistance and the metabolic syndrome are related to the severity of steatosis in the pediatric population with obesity.

BACKGROUND: To determine the factors associated with an increased risk for severe steatosis (SS) and establish the Homeostatic Model Assessment-Insulin Resistance (HOMA-IR) as a screening tool. METHODS: A cross-sectional study was performed in obese children to assess the relationship between the metabolic syndrome (MetS) and glucose metabolism alterations (GMA) and the risk for severe steatosis. RESULTS: A total of 94 children (51 males) aged from six to 14 years were included. Thirteen children (14.8%) had severe steatosis (SS). The anthropometric variables associated with SS included body mass index (BMI) (SS 34.1 vs non-SS 29.7, p = 0.005), waist circumference (cm) (100 vs 92.5, p = 0.015) and hip circumference (cm) (108 vs 100, p = 0.018). The blood parameters included alanine aminotransferase (ALT) (UI/dl) (27 vs 21, p = 0.002), gamma-glutamil transpeptidase (GGT) (UI/dl) (16 vs 15, p = 0.017), fasting glycemia (mg/dl) (96 vs 88, p = 0.006), fasting insulin (UI/dl) (25 vs 15.3, p < 0.001) and HOMA-IR score (7.1 vs 3.7, p < 0.001). Eighteen children with MetS were found to be at an increased risk for severe steatosis (odds ratio [OR] 11.36, p < 0.001). After receiver operating characteristic (ROC) curve analysis, the best area under the curve (AUC) was obtained for HOMA-R of 0.862. The HOMA-R 4.9 cut-off value had a 100% sensitivity (CI 95%: 96.2-100) and 67.9% specificity (CI 95%: 57.1-78.7) for severe steatosis. CONCLUSIONS: The presence of MetS and glucose metabolism alterations are risk factors for severe steatosis. The 4.9 cut-off value for HOMA-IR may be a risk factor for severe steatosis in obese children.

Phormidolides B and C, cytotoxic agents from the sea: enantioselective synthesis of the macrocyclic core.

New cytotoxic polyketide macrolides named phormidolides B and C were isolated from a marine sponge of the Petrosiidae family collected off the coast of Pemba (Tanzania). The isolation, structure elucidation, and enantioselective synthesis of three diastereomers of the macrocyclic core is described herein. The described synthetic methodology started from 2-deoxy-D-ribose or 2-deoxy-L-ribose and afforded the desired macrocycles with high enantiomeric purity. The key step of the synthesis is the formation of the Z-trisubstituted double bond using a Julia-Kocienski olefination. The versatility of the synthetic methodology may provide access to other enantiopure macrocycles by making changes in the starting materials or chiral inductors.

Stellatolides, a new cyclodepsipeptide family from the sponge Ecionemia acervus: isolation, solid-phase total synthesis, and full structural assignment of stellatolide A.

The marine environment is a rich source of metabolites with potential therapeutic properties and applications for humans. Here we describe the first isolation, solid-phase total synthesis, and full structural assignment of a new class of cyclodepsipeptides from the Madagascan sponge Ecionemia acervus that shows in vitro cytotoxic activities at submicromolar concentrations. Seven structures belonging to a new family of compounds, given the general name stellatolides, were characterized. The sequence and stereochemistry of all the amino acids in these molecules were established by a combination of spectroscopic analysis, chemical degradation, and derivatization studies. Furthermore, the complete structure of stellatolide A was confirmed by an efficient solid-phase method for the first total synthesis and the full structural assignment of this molecule, including the asymmetric synthesis of the unique ß-hydroxy acid moiety (Z)-3-hydroxy-6,8-dimethylnon-4-enoic acid.

Levels of active tyrosine kinase receptor determine the tumor response to Zalypsis.

BACKGROUND: Zalypsis(®) is a marine compound in phase II clinical trials for multiple myeloma, cervical and endometrial cancer, and Ewing's sarcoma. However, the determinants of the response to Zalypsis are not well known. The identification of biomarkers for Zalypsis activity would also contribute to broaden the spectrum of tumors by selecting those patients more likely to respond to this therapy. METHODS: Using in vitro drug sensitivity data coupled with a set of molecular data from a panel of sarcoma cell lines, we developed molecular signatures that predict sensitivity to Zalypsis. We verified these results in culture and in vivo xenograft studies. RESULTS: Zalypsis resistance was dependent on the expression levels of PDGFRα or constitutive phosphorylation of c-Kit, indicating that the activation of tyrosine kinase receptors (TKRs) may determine resistance to Zalypsis. To validate our observation, we measured the levels of total and active (phosphorylated) forms of the RTKs PDGFRα/ß, c-Kit, and EGFR in a new panel of diverse solid tumor cell lines and found that the IC50 to the drug correlated with RTK activation in this new panel. We further tested our predictions about Zalypsis determinants for response in vivo in xenograft models. All cells lines expressing low levels of RTK signaling were sensitive to Zalypsis in vivo, whereas all cell lines except two with high levels of RTK signaling were resistant to the drug. CONCLUSIONS: RTK activation might provide important signals to overcome the cytotoxicity of Zalypsis and should be taken into consideration in current and future clinical trials.

Isolation and structures of pipecolidepsins A and B, cytotoxic cyclic depsipeptides from the Madagascan sponge Homophymia lamellosa.

Two new cyclic depsipeptides, pipecolidepsins A and B (1 and 2), have been isolated from the sponge Homophymia lamellosa collected off the coast of Madagascar. Their structures were determined by a combination of NMR experiments and by LC-MS analysis of the amino acid fragments obtained by hydrolysis and derivatization using Marfey's reagent. In addition to several common amino acids, these peptides contain unusual residues, including 2-amino-3-hydroxy-4,5-dimethylhexanoic acid, 3-ethoxyasparagine, 3,4-dimethylglutamine, 4,7-diamino-2,3-dihydroxy-7-oxoheptanoic acid, and 3-hydroxyaspartic acid as well as a terminal 3-hydroxy-2,4,6-trimethylheptanoic acid residue. Pipecolidepsins A and B displayed cytotoxic activity against a panel of different human tumor cell lines.

Tanjungides A and B: new antitumoral bromoindole derived compounds from Diazona cf formosa. isolation and total synthesis.

Tanjungides A (1) (Z isomer) and B (2) (E isomer), two novel dibrominated indole enamides, have been isolated from the tunicate Diazona cf formosa. Their structures were determined by spectroscopic methods including HRMS, and extensive 1D and 2D NMR. The stereochemistry of the cyclised cystine present in both compounds was determined by Marfey's analysis after chemical degradation and hydrolysis. We also report the first total synthesis of these compounds using methyl 1H-indole-3-carboxylate as starting material and a linear sequence of 11 chemical steps. Tanjungides A and B exhibit significant cytotoxicity against human tumor cell lines.

PM534, an Optimized Target-Protein Interaction Strategy through the Colchicine Site of Tubulin.

Targeting microtubules is the most effective wide-spectrum pharmacological strategy in antitumoral chemotherapy, and current research focuses on reducing main drawbacks: neurotoxicity and resistance. PM534 is a novel synthetic compound derived from the Structure-Activity-Relationship study on the natural molecule PM742, isolated from the sponge of the order Lithistida, family Theonellidae, genus Discodermia (du Bocage 1869). PM534 targets the entire colchicine binding domain of tubulin, covering four of the five centers of the pharmacophore model. Its nanomolar affinity and high retention time modulate a strikingly high antitumor activity that efficiently overrides two resistance mechanisms in cells (detoxification pumps and tubulin ßIII isotype overexpression). Furthermore, PM534 induces significant inhibition of tumor growth in mouse xenograft models of human non-small cell lung cancer. Our results present PM534, a highly effective new compound in the preclinical evaluation that is currently in its first human Phase I clinical trial.

Efficacy and safety of a structured de-escalation from antipseudomonal ß-lactams in bloodstream infections due to Enterobacterales (SIMPLIFY): an open-label, multicentre, randomised trial.

BACKGROUND: De-escalation from broad-spectrum to narrow-spectrum antibiotics is considered an important measure to reduce the selective pressure of antibiotics, but a scarcity of adequate evidence is a barrier to its implementation. We aimed to determine whether de-escalation from an antipseudomonal ß-lactam to a narrower-spectrum drug was non-inferior to continuing the antipseudomonal drug in patients with Enterobacterales bacteraemia. METHODS: An open-label, pragmatic, randomised trial was performed in 21 Spanish hospitals. Patients with bacteraemia caused by Enterobacterales susceptible to one of the de-escalation options and treated empirically with an antipseudomonal ß-lactam were eligible. Patients were randomly assigned (1:1; stratified by urinary source) to de-escalate to ampicillin, trimethoprim-sulfamethoxazole (urinary tract infections only), cefuroxime, cefotaxime or ceftriaxone, amoxicillin-clavulanic acid, ciprofloxacin, or ertapenem in that order according to susceptibility (de-escalation group), or to continue with the empiric antipseudomonal ß-lactam (control group). Oral switching was allowed in both groups. The primary outcome was clinical cure 3-5 days after end of treatment in the modified intention-to-treat (mITT) population, formed of patients who received at least one dose of study drug. Safety was assessed in all participants. Non-inferiority was declared when the lower bound of the 95% CI of the absolute difference in cure rate was above the -10% non-inferiority margin. This trial is registered with EudraCT (2015-004219-19) and ClinicalTrials.gov (NCT02795949) and is complete. FINDINGS: 2030 patients were screened between Oct 5, 2016, and Jan 23, 2020, of whom 171 were randomly assigned to the de-escalation group and 173 to the control group. 164 (50%) patients in the de-escalation group and 167 (50%) in the control group were included in the mITT population. 148 (90%) patients in the de-escalation group and 148 (89%) in the control group had clinical cure (risk difference 1·6 percentage points, 95% CI -5·0 to 8·2). The number of adverse events reported was 219 in the de-escalation group and 175 in the control group, of these, 53 (24%) in the de-escalation group and 56 (32%) in the control group were considered severe. Seven (5%) of 164 patients in the de-escalation group and nine (6%) of 167 patients in the control group died during the 60-day follow-up. There were no treatment-related deaths. INTERPRETATION: De-escalation from an antipseudomonal ß-lactam in Enterobacterales bacteraemia following a predefined rule was non-inferior to continuing the empiric antipseudomonal drug. These results support de-escalation in this setting. FUNDING: Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases; Spanish Clinical Research and Clinical Trials Platform, co-financed by the EU; European Development Regional Fund "A way to achieve Europe", Operative Program Intelligence Growth 2014-2020.