Molecular and therapeutic insights of rapamycin: a multi-faceted drug from Streptomyces hygroscopicus.

The advancement in pharmaceutical research has led to the discovery and development of new combinatorial life-saving drugs. Rapamycin is a macrolide compound produced from Streptomyces hygroscopicus. Rapamycin and its derivatives are one of the promising sources of drug with broad spectrum applications in the medical field. In recent times, rapamycin has gained significant attention as of its activity against cytokine storm in COVID-19 patients. Rapamycin and its derivatives have more potency when compared to other prevailing drugs. Initially, it has been used exclusively as an anti-fungal drug. Currently rapamycin has been widely used as an immunosuppressant. Rapamycin is a multifaceted drug; it has anti-cancer, anti-viral and anti-aging potentials. Rapamycin has its specific action on mTOR signaling pathway. mTOR has been identified as a key regulator of different pathways. There will be an increased demand for rapamycin, because it has lesser adverse effects when compared to steroids. Currently researchers are focused on the production of effective rapamycin derivatives to combat the growing demand of this wonder drug. The main focus of the current review is to explore the origin, development, molecular mechanistic action, and the current therapeutic aspects of rapamycin. Also, this review article revealed the potential of rapamycin and the progress of rapamycin research. This helps in understanding the exact potency of the drug and could facilitate further studies that could fill in the existing knowledge gaps. The study also gathers significant data pertaining to the gene clusters and biosynthetic pathways involved in the synthesis and production of this multi-faceted drug. In addition, an insight into the mechanism of action of the drug and important derivatives of rapamycin has been expounded. The fillings of the current review, aids in understanding the underlying molecular mechanism, strain improvement, optimization and production of rapamycin derivatives.

Head and neck mycetoma: Clinical findings, investigations, and predictors for recurrence of the disease in Sudan: A retrospective study.

INTRODUCTION: Mycetoma is a unique neglected tropical disease which is found endemic in areas known as the "mycetoma belt". Head and neck mycetoma is a rarity and it has many devastating impacts on patients and communities. In this study, we assessed clinical findings, investigations, and predictors for recurrence of head and neck mycetoma in Sudan. METHODOLOGY: A retrospective study was conducted at Mycetoma Research Center in Khartoum between January 1999 and December 2020 for all patients with head and neck mycetoma. Data were analyzed using R software version 4.0.2. RESULTS: We included 107 patients with head and neck mycetoma. 65.4% were young adult males from mycetoma endemic areas in Sudan, and most of them were students (33.6%). Most of patients (64.4%) had actinomycetoma. Before presenting with head and neck mycetoma, majority (75.7%) had a long duration with mycetoma, and 30.8% had a history of trauma. The commonest invaded site was the parietal region (30.8%). The lesion started gradually in most of the patients (96.3%). 53.3% of the patients had large size lesions with no sweating, regional lymph nodes involvement, or distal vein involvement. CT scan was the most accurate diagnostic tool while 8.4% of patients were diagnosed by clinical examinations only. Laboratory investigations confirmed that 24/45 (44.4%) of actinomycetoma was caused by Streptomyces somaliensis while 13/28 (46.4%) of eumycetoma was caused by Madurella mycetomatis. All patients with recurrence of head and neck mycetoma underwent surgical excision of the lesion (n = 41/41 {100%}, p < 0.001). CONCLUSION: In head and neck mycetoma, the most common type was actinomycetes in Sudan. Majority had a long course of mycetoma and the commonest causative organism was Streptomyces somaliensis. The treatment outcome was poor and characterized by a low cure rate.

Dihydromaniwamycin E, a Heat-Shock Metabolite from Thermotolerant Streptomyces sp. JA74, Exhibiting Antiviral Activity against Influenza and SARS-CoV-2 Viruses.

Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50 values of 25.7 and 63.2 µM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50 values (for infection of 293TA cells) of 19.7 and 9.7 µM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50 concentrations.

Investigation of Streptomyces sp. Strain EMB24 Secondary Metabolite Profile Has Unraveled Its Extraordinary Antibacterial Potency Against Drug-Resistant Bacteria.

With the overuse and misuse of antibiotics amid COVID-19 pandemic, the antimicrobial resistance, which is already a global challenge, has accelerated its pace significantly. Finding novel and potential antibiotics seems one of the probable solutions. In this work, a novel Streptomyces sp. strain EMB24 was isolated and found to be an excellent source of antimicrobials as confirmed by agar-plug assay. It showed antibacterial activity against infection-causing bacteria, namely Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. In addition, Streptomyces sp. strain EMB24 inhibited the growth methicillin-resistant Staphylococcus aureus (MRSA), tetracycline-resistant Neisseria gonorrhoeae, and ampicillin-resistant Neisseria gonorrhoeae. Furthermore, to get deep insights about the genome and biosynthetic gene clusters producing antibiotics, whole genome sequencing was done. The strain EMB24 is closely related to the Streptomyces longispororuber as revealed by phylogenetic analysis which is a potential source of antibiotics and pigments as undecylprodigiosin and metacycloprodigiosin belonging to the class prodigiosin. Naphthyridinomycin, alkylresorcinols, desferrioxamine B and E, venezuelin, aborycin, MS-271, and siamycin are potent therapeutics that shared 100% similarity with the reference strain as revealed by the online antiSMASH tool.

Streptomyces BAC Cloning of a Large-Sized Biosynthetic Gene Cluster of NPP B1, a Potential SARS-CoV-2 RdRp Inhibitor.

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

Bhimamycin J, a Rare Benzo[f]isoindole-dione Alkaloid from the Marine-Derived Actinomycete Streptomyces sp. MS180069.

Chemical investigation on a Streptomyces sp. strain MS180069 isolated from a sediment sample collected from the South China Sea, yielded the new benzo[f]isoindole-dione alkaloid, bhimamycin J (1). The structure was determined by extensive spectroscopic analysis, including HRMS, 1D, 2D NMR, and X-ray diffraction techniques. A molecular docking study revealed 1 as a new molecular motif that binds with human angiotensin converting enzyme2 (ACE2), recently described as the cell surface receptor responsible for uptake of 2019-CoV-2. Using enzyme assays we confirm that 1 inhibits human ACE2 79.7 % at 25 µg/mL.

Reconstitution of a mini-gene cluster combined with ribosome engineering led to effective enhancement of salinomycin production in Streptomyces albus.

Salinomycin, an FDA-approved polyketide drug, was recently identified as a promising anti-tumour and anti-viral lead compound. It is produced by Streptomyces albus, and the biosynthetic gene cluster (sal) spans over 100 kb. The genetic manipulation of large polyketide gene clusters is challenging, and approaches delivering reliable efficiency and accuracy are desired. Herein, a delicate strategy to enhance salinomycin production was devised and evaluated. We reconstructed a minimized sal gene cluster (mini-cluster) on pSET152 including key genes responsible for tailoring modification, antibiotic resistance, positive regulation and precursor supply. These genes were overexpressed under the control of constitutive promoter PkasO* or Pneo . The pks operon was not included in the mini-cluster, but it was upregulated by SalJ activation. After the plasmid pSET152::mini-cluster was introduced into the wild-type strain and a chassis host strain obtained by ribosome engineering, salinomycin production was increased to 2.3-fold and 5.1-fold compared with that of the wild-type strain respectively. Intriguingly, mini-cluster introduction resulted in much higher production than overexpression of the whole sal gene cluster. The findings demonstrated that reconstitution of sal mini-cluster combined with ribosome engineering is an efficient novel approach and may be extended to other large polyketide biosynthesis.

Discovery of anti-infective adipostatins through bioactivity-guided isolation and heterologous expression of a type III polyketide synthase.

Antibiotic resistance and emerging viral pandemics have posed an urgent need for new anti-infective drugs. By screening our microbial extract library against the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the notorious ESKAPE pathogens, an active fraction was identified and purified, leading to an initial isolation of adipostatins A (1) and B (2). In order to diversify the chemical structures of adipostatins toward enhanced biological activities, a type III polyketide synthase was identified from the native producer, Streptomyces davawensis DSM101723, and was subsequently expressed in an E. coli host, resulting in the isolation of nine additional adipostatins 3-11, including two new analogs (9 and 11). The structures of 1-11 were established by HRMS, NMR, and chemical derivatization, including using a microgram-scale meta-chloroperoxybenzoic acid epoxidation-MS/MS analysis to unambiguously determine the double bond position in the alkyl chain. The present study discovered SARS-CoV-2 main protease inhibitory activity for the class of adipostatins for the first time. Several of the adipostatins isolated also exhibited antimicrobial activity against selected ESKAPE pathogens.

Antiviral Bafilomycins from a Feces-Inhabiting Streptomyces sp.

A new bafilomycin derivative (1) and another seven known bafilomycins (2-8) were isolated from feces-derived Streptomyces sp. HTL16. The structure of 1 was elucidated by 1D and 2D NMR spectroscopic analysis. Biological testing demonstrated that these bafilomycins exhibited potent antiviral activities against the influenza A and SARS-CoV-2 viruses, with IC50 values in the nanomolar range, by inhibiting the activity of endosomal ATP-driven proton pumps.

Ivermectin Docks to the SARS-CoV-2 Spike Receptor-binding Domain Attached to ACE2.

BACKGROUND/AIM: Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One drug that has attracted interest is the antiparasitic compound ivermectin, a macrocyclic lactone derived from the bacterium Streptomyces avermitilis. We carried out a docking study to determine if ivermectin might be able to attach to the SARS-CoV-2 spike receptor-binding domain bound with ACE2. MATERIALS AND METHODS: We used the program AutoDock Vina Extended to perform the docking study. RESULTS: Ivermectin docked in the region of leucine 91 of the spike and histidine 378 of the ACE2 receptor. The binding energy of ivermectin to the spike-ACE2 complex was -18 kcal/mol and binding constant was 5.8 e-08. CONCLUSION: The ivermectin docking we identified may interfere with the attachment of the spike to the human cell membrane. Clinical trials now underway should determine whether ivermectin is an effective treatment for SARS-Cov2 infection.