Changing Biosynthetic Profiles by Expressing bldA in Streptomyces Strains

Recently we described an unusual way of activating a cryptic gene cluster when we explored the origin of the bald phenotype of Streptomyces calvus. Complementation of S. calvus with a correct copy of bldA restored sporulation and additionally promoted production of a new natural products. In this study we report on the expression of bldA in several Streptomyces strains that have been described as “poorly sporulating” strains. In seven out of 15 cases, HPLC profiling revealed the production of new compounds, and in two cases the overproduction of known compounds. Two compounds were isolated and their structures were determined.     

Identification and Characterization of the Streptazone E Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08

Streptazone derivatives isolated from Streptomyces species are piperidine alkaloids with a cyclopenta[b]pyridine scaffold. Previous studies indicated that these compounds are polyketides, but the biosynthetic enzymes responsible for their synthesis are unknown. Here, we have identified the streptazone E biosynthetic gene cluster in Streptomyces sp. MSC090213JE08, which encodes a modular type I PKS and tailoring enzymes that include an aminotransferase, three oxidoreductases, and two putative cyclases. The functions of the six tailoring enzymes were analyzed by gene disruption, and two putative biosynthetic intermediates that accumulated in particular mutants were structurally elucidated. On the basis of these results, we propose a pathway for the biosynthesis of streptazone E in which the two putative cyclases of the nuclear transport factor 2–like superfamily are responsible for C?C bond formation coupled with epoxide ring opening to give the five‐membered ring of streptazone E.     

Streptocollin,a Type IV Lanthipeptide Produced by Streptomyces collinus Tü 365

Lanthipeptides are ribosomally synthesized and post‐translationally modified microbial secondary metabolites. Here, we report the identification and isolation of streptocollin from Streptomyces collinus Tü 365, a new member of class IV lanthipeptides. Insertion of the constitutive ermE* promoter upstream of the lanthipeptide synthetase gene stcL resulted in peptide production. The streptocollin gene cluster was heterologously expressed in S. coelicolor M1146 and M1152 with 3.5‐ and 5.5‐fold increased yields, respectively. The structure and ring topology of streptocollin were determined by high resolution MS/MS analysis. Streptocollin contains four macrocyclic rings, with one lanthionine and three methyllanthionine residues. To the best of our knowledge, this is the first report on the isolation of a class IV lanthipeptide in preparative amounts, and on the successful heterologous expression of a class IV lanthipeptide gene cluster.     

Discovery of a Highly Selective PLD2 Inhibitor (ML395): A New Probe with Improved Physiochemical Properties and Broad‐Spectrum Antiviral Activity against Influenza Strains

Further chemical optimization of the halopemide‐derived family of dual phospholipase D1/2 (PLD1/2) inhibitors afforded ML395 (VU0468809), a potent, >80‐fold PLD2 selective allosteric inhibitor (cellular PLD1, IC₅₀>30 000 nM ; cellular PLD2, IC₅₀=360 nM ). Moreover, ML395 possesses an attractive in vitro DMPK profile, improved physiochemical properties, ancillary pharmacology (Eurofins Panel) cleaner than any other reported PLD inhibitor, and has been found to possess interesting activity as an antiviral agent in cellular assays against a range of influenza strains (H1, H3, H5 and H7).     

Aromatic Polyketide GTRI‐02 is a Previously Unidentified Product of the act Gene Cluster in Streptomyces coelicolor A3(2)

The biosynthesis of aromatic polyketides derived from type II polyketide synthases (PKSs) is complex, and it is not uncommon that highly similar gene clusters give rise to diverse structural architectures. The act biosynthetic gene cluster (BGC) of the model actinomycete Streptomyces coelicolor A3(2) is an archetypal type II PKS. Here we show that the act BGC also specifies the aromatic polyketide GTRI‐02 ( 1 ) and propose a mechanism for the biogenesis of its 3,4‐dihydronaphthalen‐1(2H)‐one backbone. Polyketide 1 was also produced by Streptomyces sp. MBT76 after activation of the act‐like qin gene cluster by overexpression of the pathway‐specific activator. Mining of this strain also identified dehydroxy‐GTRI‐02 ( 2 ), which most likely originated from dehydration of 1 during the isolation process. This work shows that even extensively studied model gene clusters such as act of S. coelicolor can still produce new chemistry, offering new perspectives for drug discovery.     

Characterisation of acorn oils extracted by hexane and by supercritical carbon dioxide

Acorn fruit oils from two species of oak, Quercus rotundifolia L. (holm‐oak) and Quercus suber L. (cork‐oak), were extracted by n‐hexane. The acorn fruit of Quercus rotundifolia L. was also extracted by supercritical CO₂ at 18 MPa and 313 K, a superficial velocity of 2.5 × 10^?4 ms^?1, and a particle size diameter of 2.7 × 10^?4 m. The oils were characterised in terms of fatty acids, triglycerides, sterols, tocopherols, and phospholipids. The main fatty acid in both fruit species was oleic acid (about 65%), followed by linoleic acid (about 16.5–17%) and palmitic acid (about 12.1–13.4%). The main triglyceride found in acorn oils was the OOO (oleic, oleic, oleic) triglyceride (33–38%), followed by the POO (palmitic, oleic, oleic) triglyceride (12.6–18.2%). In terms of sterols, the main component in acorn oils of both species was β‐sitosterol (83.5–89%), followed by stigmasterol (about 3%). However, in Quercus suber L., acorn oil was found to consist to 10.2% of campesterol. The amount of cholesterol was low (0.27% for the Quercus rotundifolia L. oil extracted by supercritical fluid extraction, and 0.18% for the oil extracted by n‐hexane). The Quercus suber L. acorn oil presented 0.1% of cholesterol. The total amount of tocopherols in Quercus rotundifolia L. acorn oils was almost the same when the oil was extracted by n‐hexane (973 mg/kg oil) or by supercritical CO₂ (1006 mg/kg oil). The Quercus suber L. acorn oil presented a high value of total tocopherols (1486 mg/kg oil). The supercritical CO₂ did not extract the phospholipids. The amount of phospholipids was very similar for both species of oak acorn oils extracted by n‐hexane. Oxidative stability was also studied, by using the peroxide value and the Rancimat method, revealing that all the oils were significantly protected against oxidation. The influence of storage, under several conditions, on the oxidative stability was also studied. The Quercus rotundifolia L. oil extracted by n‐hexane was better protected against oxidation after a few days of storage at 60 °C.     

In Vivo Delivery and Activation of Masked Fluorogenic Hydrolase Substrates by Endogenous Hydrolases in C. elegans

Protein expression and localization are often studied in vivo by tagging molecules with green fluorescent protein (GFP), yet subtle changes in protein levels are not easily detected. To develop a sensitive in vivo method to amplify fluorescence signals and allow cell‐specific quantification of protein abundance changes, we sought to apply an enzyme‐activated cellular fluorescence system in vivo by delivering ester‐masked fluorophores to Caenorhabditis elegans neurons expressing porcine liver esterase (PLE). To aid uptake into sensory neuron membranes, we synthesized two novel fluorogenic hydrolase substrates with long hydrocarbon tails. Recombinant PLE activated these fluorophores in vitro. In vivo activation occurred in sensory neurons, along with potent activation in intestinal lysosomes quantifiable by imaging and microplate and partially attributable to gut esterase 1 (GES‐1) activity. These data demonstrate the promise of biorthogonal hydrolases and their fluorogenic substrates as in vivo neuronal imaging tools and for characterizing endogenous C. elegans hydrolase substrate specificities.     

A Dual‐Targeting Octaguanidine–Doxorubicin Conjugate Transporter for Inducing Caspase‐Mediated Apoptosis on Folate‐Expressing Cancer Cells

An efficient synthetic framework was assembled (G8‐FKE‐FA‐Dox), consisting of a lysosome‐targeting octaguanidine molecular transporter with a cathepsin B (cath B)‐specific peptide substrate, folic acid, and the potent chemotherapeutic drug doxorubicin (Dox). Because the folate receptor (FR) and cath B are overexpressed in malignant cells, this transporter conjugate successfully executed lysosome‐mediated transport of Dox to FR‐positive tumor cells, illustrating this framework as an excellent targeted drug delivery system (TDDS). G8‐FKE‐FA‐Dox was shown to exhibit selective toxicity toward FR‐overexpressing cancer cells, with an IC₅₀ value superior to that of the USFDA‐approved Lipodox^TM and proportional to that of free Dox via selective induction of apoptosis by the activation of caspases 8, 9, and 3. This TDDS was observed to be nontoxic to red blood cells and lymphocytes at neutral pH. Furthermore the tumor‐targeting dissemination pattern of this system was revealed by monitoring the in vivo biodistribution of the carrier (G8‐FKE‐FA‐FL) in normal and FR‐overexpressing tumor‐bearing mice.     

Trastuzumab Deruxtecan: Changing the Destiny of HER2 Expressing Solid Tumors

HER2 targeted therapies have significantly improved prognosis of HER2-positive breast and gastric cancer. HER2 overexpression and mutation is the pathogenic driver in non-small cell lung cancer (NSCLC) and colorectal cancer, however, to date, there are no approved HER2-targeted therapies with these indications. Trastuzumab deruxtecan (T-DXd) is a novel HER2-directed antibody drug conjugate showing significant anti-tumor activity in heavily pre-treated HER2-positive breast and gastric cancer patients. Preliminary data have shown promising objective response rates in patients with HER2-positive NSCLC and colorectal cancer. T-DXd has an acceptable safety profile, however with concerns regarding potentially serious treatment-emergent adverse events. In this review we focus on the pharmacologic characteristics and toxicity profile of T-Dxd, and provide an update on the most recent results of clinical trials of T-DXd in solid tumors. The referenced papers were selected through a PubMed search performed on 16 March 2021 with the following searching terms: T-DXd and breast cancer, or gastric cancer, or non-small cell lung cancer (NSCLC), or colorectal cancer. Oral presentation, abstracts, and posters presented at the American Society of Clinical Oncology (ASCO, Alexandria, VA, USA) 2020 and the European Society for Medical Oncology (ESMO, Lugano, Switzerland) 2020 annual meetings were retrieved for data on T-DXd. We also overview ongoing research and data of combination therapies currently under investigation, which will impact on future therapeutic strategies. Clinicaltrials.gov was searched to identify ongoing clinical trials of T-DXd alone or in combination in solid tumors.     

Antibody Epitope of Human α‐Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease

α‐Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α‐galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α‐galactosidase A is known as Fabry disease or Fabry–Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy‐limiting, and eventually life‐threatening complications of ERT. The present study focused on the epitope determination of human α‐galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309–332) recognized by a human monoclonal anti‐αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309–332), was synthesized by solid‐phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (K_D=39×10^?9 m ), which is nearly identical to that of the full‐length enzyme (K_D=16×10^?9 m ). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full‐length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.     

Characterization of Enzymes Catalyzing Transformations of Cysteine S‐Conjugated Intermediates in the Lincosamide Biosynthetic Pathway

Lincosamides such as lincomycin A, celesticetin, and Bu‐2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l ‐proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5′‐phosphate‐dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S‐conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C?S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu‐2545. The substrates used in these studies are the β‐anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved.     

The ɛ‐Amino Group of Protein Lysine Residues Is Highly Susceptible to Nonenzymatic Acylation by Several Physiological Acyl‐CoA Thioesters

Mitochondrial enzymes implicated in the pathophysiology of diabetes, cancer, and metabolic syndrome are highly regulated by acetylation. However, mitochondrial acetyltransferases have not been identified. Here, we show that acetylation and also other acylations are spontaneous processes that depend on pH value, acyl‐CoA concentration and the chemical nature of the acyl residue. In the case of a peptide derived from carbamoyl phosphate synthetase 1, the rates of succinylation and glutarylation were up to 150 times than for acetylation. These results were confirmed by using the protein substrate cyclophilin A (CypA). Deacylation experiments revealed that SIRT3 exhibits deacetylase activity but is not able to remove any of the succinyl groups from CypA, whereas SIRT5 is an effective protein desuccinylase. Thus, the acylation landscape on lysine residues might largely depend on the enzymatic activity of specific sirtuins, and the availability and reactivity of acyl‐CoA compounds.     

Antivirulence Isoquinolone Mannosides: Optimization of the Biaryl Aglycone for FimH Lectin Binding Affinity and Efficacy in the Treatment of Chronic UTI

Uropathogenic E. coli (UPEC) employ the mannose‐binding adhesin FimH to colonize the bladder epithelium during urinary tract infection (UTI). Previously reported FimH antagonists exhibit good potency and efficacy, but low bioavailability and a short half‐life in vivo. In a rational design strategy, we obtained an X‐ray structure of lead mannosides and then designed mannosides with improved drug‐like properties. We show that cyclizing the carboxamide onto the biphenyl B‐ring aglycone of biphenyl mannosides into a fused heterocyclic ring, generates new biaryl mannosides such as isoquinolone 22 (2‐methyl‐4‐(1‐oxo‐1,2‐dihydroisoquinolin‐7‐yl)phenyl α‐d ‐mannopyranoside) with enhanced potency and in vivo efficacy resulting from increased oral bioavailability. N‐Substitution of the isoquinolone aglycone with various functionalities produced a new potent subseries of FimH antagonists. All analogues of the subseries have higher FimH binding affinity than unsubstituted lead 22 , as determined by thermal shift differential scanning fluorimetry assay. Mannosides with pyridyl substitution on the isoquinolone group inhibit bacteria‐mediated hemagglutination and prevent biofilm formation by UPEC with single‐digit nanomolar potency, which is unprecedented for any FimH antagonists or any other antivirulence compounds reported to date.     

Fluorine‐Containing 6,7‐Dialkoxybiaryl‐Based Inhibitors for Phosphodiesterase 10 A: Synthesis and in vitro Evaluation of Inhibitory Potency,Selectivity, and Metabolism

Based on the potent phosphodiesterase 10 A (PDE10A) inhibitor PQ‐10, we synthesized 32 derivatives to determine relationships between their molecular structure and binding properties. Their roles as potential positron emission tomography (PET) ligands were evaluated, as well as their inhibitory potency toward PDE10A and other PDEs, and their metabolic stability was determined in vitro. According to our findings, halo‐alkyl substituents at position 2 of the quinazoline moiety and/or halo‐alkyloxy substituents at positions 6 or 7 affect not only the compounds′ affinity, but also their selectivity toward PDE10A. As a result of substituting the methoxy group for a monofluoroethoxy or difluoroethoxy group at position 6 of the quinazoline ring, the selectivity for PDE10A over PDE3A increased. The same result was obtained by 6,7‐difluoride substitution on the quinoxaline moiety. Finally, fluorinated compounds (R)‐7‐(fluoromethoxy)‐6‐methoxy‐4‐(3‐(quinoxaline‐2‐yloxy)pyrrolidine‐1‐yl)quinazoline ( 16 a ), 19 a – d , (R)‐tert‐butyl‐3‐(6‐fluoroquinoxalin‐2‐yloxy)pyrrolidine‐1‐carboxylate ( 29 ), and 35 (IC₅₀ PDE10A 11–65 nM ) showed the highest inhibitory potential. Further, fluoroethoxy substitution at position 7 of the quinazoline ring improved metabolic stability over that of the lead structure PQ‐10.     

Characterization of Mono- and Bi-Transgenic Pig-Derived Epidermal Keratinocytes Expressing Human FUT2 and GLA Genes—In Vitro Studies

Pig-to-human xenotransplantation seems to be the response to the contemporary shortage of tissue/organ donors. Unfortunately, the phylogenetic distance between pig and human implies hyperacute xenograft rejection. In this study, we tested the hypothesis that combining expression of human α1,2-fucosyltransferase (hFUT2) and α-galactosidase A (hGLA) genes would allow for removal of this obstacle in porcine transgenic epidermal keratinocytes (PEKs). We sought to determine not only the expression profiles of recombinant human α1,2-fucosyltransferase (rhα1,2-FT) and α-galactosidase A (rhα-Gal A) proteins, but also the relative abundance (RA) of Galα1→3Gal epitopes in the PEKs stemming from not only hFUT2 or hGLA single-transgenic and hFUT2×hGLA double-transgenic pigs. Our confocal microscopy and Western blotting analyses revealed that both rhα1,2-FT and rhα-Gal A enzymes were overabundantly expressed in respective transgenic PEK lines. Moreover, the semiquantitative levels of Galα1→3Gal epitope that were assessed by lectin fluorescence and lectin blotting were found to be significantly diminished in each variant of genetically modified PEK line as compared to those observed in the control nontransgenic PEKs. Notably, the bi-transgenic PEKs were characterized by significantly lessened (but still detectable) RAs of Galα1→3Gal epitopes as compared to those identified for both types of mono-transgenic PEK lines. Additionally, our current investigation showed that the coexpression of two protective transgenes gave rise to enhanced abrogation of Galα→3Gal epitopes in hFUT2×hGLA double-transgenic PEKs. To summarize, detailed estimation of semiquantitative profiles for human α-1,2-FT and α-Gal A proteins followed by identification of the extent of abrogating the abundance of Galα1→3Gal epitopes in the ex vivo expanded PEKs stemming from mono- and bi-transgenic pigs were found to be a sine qua non condition for efficiently ex situ protecting stable lines of skin-derived somatic cells inevitable in further studies. The latter is due to be focused on determining epigenomic reprogrammability of single- or double-transgenic cell nuclei inherited from adult cutaneous keratinocytes in porcine nuclear-transferred oocytes and corresponding cloned embryos. To our knowledge, this concept was shown to represent a completely new approach designed to generate and multiply genetically transformed pigs by somatic cell cloning for the needs of reconstructive medicine and dermoplasty-mediated tissue engineering of human integumentary system.     

N‐Benzyl Substitution of Polyhydroxypyrrolidines: The Way to Selective Inhibitors of Golgi α‐Mannosidase II

Inhibition of the biosynthesis of complex N‐glycans in the Golgi apparatus influences progress of tumor growth and metastasis. Golgi α‐mannosidase II (GMII) has become a therapeutic target for drugs with anticancer activities. One critical task for successful application of GMII drugs in medical treatments is to decrease their unwanted co‐inhibition of lysosomal α‐mannosidase (LMan), a weakness of all known potent GMII inhibitors. A series of novel N‐substituted polyhydroxypyrrolidines was synthesized and tested with modeled GH38 α‐mannosidases from Drosophila melanogaster (GMIIb and LManII). The most potent structures inhibited GMIIb (K_i=50–76 μm , as determined by enzyme assays) with a significant selectivity index of IC₅₀(LManII)/IC₅₀(GMIIb) >100. These compounds also showed inhibitory activities in in vitro assays with cancer cell lines (leukemia, IC₅₀=92–200 μm ) and low cytotoxic activities in normal fibroblast cell lines (IC₅₀>200 μm ). In addition, they did not show any significant inhibitory activity toward GH47 Aspergillus saitoiα1,2‐mannosidase. An appropriate stereo configuration of hydroxymethyl and benzyl functional groups on the pyrrolidine ring of the inhibitor may lead to an inhibitor with the required selectivity for the active site of a target α‐mannosidase.