Teleocidin-producing genotype of Streptomyces clavuligerus ATCC 27064.

Streptomyces clavuligerus is an industrially important producer of clavulanic acid (CA), a ß-lactamase inhibitor which is used together with amoxicillin in one of the most widely prescribed antibacterial medicines, the co-amoxiclav. In a mid-eighties ATCC vial of S. clavuligerus ATCC 27064 culture, we have found a new genotype, which was apparently lost from the subsequent ATCC collection stocks, and has remained obscure to the scientific community. Most importantly, this genotype harbors teleocidin (lyngbyatoxin) biosynthetic genes, which are located on an enigmatic 138 kb chromosomal region and support accumulation of significant amounts of these highly toxic, tumor-promoting secondary metabolites in cultures of S. clavuligerus. While this genomic region is completely absent from all published sequences for S. clavuligerus ATCC strain, at least one of the industrial strains for commercial production of CA, originating from ATCC 27064, retained the genetic potential for production of teleocidins. The origin of teleocidin biosynthetic cluster can now be traced back to early S. clavuligerus stocks at the ATCC. Our work provides a genome sequence and a deposited monoisolate of this genotype. Given the scale of industrial use of S. clavuligerus world-wide and toxicity of teleocidins, we also discuss the environmental and safety implications and provide a method of abolishing teleocidin production without affecting productivity of CA. KEY POINTS: • Early stocks of S. clavuligerus ATCC 27064 produce toxic teleocidins • Teleocidin biosynthetic genes were found within a distinct S. clavuligerus genotype • The genotype has been passed on to some industrial clavulanic acid producer strains.

Understanding of cabotegravir degradation through isolation and characterization of key degradation products and kinetic studies.

Cabotegravir is a novel human immunodeficiency virus integrase enzyme inhibitor used for prevention and treatment of HIV infection. The combinational final dosage form, as extended release injection suspension in combination with rilpivirine and as cabotegravir tablets (for lead-in therapy), was recently approved in Canada, EU and in USA and is currently seeking approval also in other countries. The subject of this investigation was to study the degradation of cabotegravir under different stress conditions as per the International Council for Harmonization (ICH) guidelines. The drug substance was found to be stable in thermal, photolytic and basic stress conditions, but degraded under acidic and oxidative stress conditions. It was determined that four main degradation products of cabotegravir are formed in forced degradation studies. All four main degradation products were isolated using preparative chromatography and subjected to NMR and HRMS analysis in order to determine their structure. We proposed degradation pathways of cabotegravir under acidic stress conditions in solution based on the structure of isolated degradation products, cabotegravir degradation kinetic studies and degradation studies on two isolated key degradation products. Moreover, degradation pathway to predominant oxidation degradation product is proposed based on the adduct of cabotegravir and peroxide species, which was identified by LC-HRMS analysis. This is the first report to the best of our knowledge that describes characterized cabotegravir forced degradation impurities and provides insights into its degradation pathways.

On the Stability and Degradation Pathways of Venetoclax under Stress Conditions.

Venetoclax is an orally bioavailable, B-cell lymphoma-2 (BCL-2) selective inhibitor, used for the treatment of various types of blood cancers, such as chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL). In this study we investigated the degradation of venetoclax under various stress conditions including acidic, basic, oxidative, photolytic and thermolytic conditions. We isolated and identified six of its main degradation products produced in forced degradation studies. The structures of the isolated degradation products were determined by using nuclear magnetic resonance (NMR) spectroscopy, high resolution mass spectrometry (HRMS) and infrared (IR) spectroscopy. Additionally, one oxidation degradation product was identified with comparison to a commercially obtained venetoclax impurity. We proposed the key degradation pathways of venetoclax in solution. To the best of our knowledge, no structures of degradation products of venetoclax have been previously published. The study provides novel and primary knowledge of the stability characteristics of venetoclax under stress conditions. Venetoclax is currently the only BCL-2 protein inhibitor on the market. In addition to single agent treatment, it is effective in combinational therapy, so future drug development involving venetoclax can be expected. A better insight into the stability properties of the therapeutic can facilitate future studies involving venetoclax and aid in the search of new similar therapeutics.

Identification of mixed di-cation forms of G-quadruplex in solution.

Multinuclear NMR study has demonstrated that G-quadruplex adopted by d(G3T4G4) exhibits two cation binding sites between three of its G-quartets. Titration of tighter binding K+ ions into the solution of d(G3T4G4)2 folded in the presence of 15NH4+ ions uncovered a mixed mono-K+-mono-15NH4+ form that represents intermediate in the conversion of di-15NH4+ into di-K+ form. Analogously, 15NH4+ ions were found to replace Na+ ions inside d(G3T4G4)2 quadruplex. The preference of 15NH4+ over Na+ ions for the two binding sites is considerably smaller than the preference of K+ over 15NH4+ ions. The two cation binding sites within the G-quadruplex core differ to such a degree that 15NH4+ ions bound to the site, which is closer to the edge-type loop, are always replaced first during titration by K+ ions. The second binding site is not taken up by K+ ion until K+ ion already resides at the first binding site. Quantitative analysis of concentrations of the three di-cation forms, which are in slow exchange on the NMR time scale, at 12 K+ ion concentrations afforded equilibrium binding constants. K+ ion binding to sites U and L within d(G3T4G4)2 is more favorable with respect to 15NH4+ ions by Gibbs free energies of approximately -24 and -18 kJ mol(-1) which includes differences in cation dehydration energies, respectively.

d(G3T4G4) forms unusual dimeric G-quadruplex structure with the same general fold in the presence of K+, Na+ or NH4+ ions.

We have recently communicated that DNA oligonucleotide d(G(3)T(4)G(4)) forms a dimeric G-quadruplex in the presence of K(+) ions [J. Am. Chem. Soc.2003, 125, 7866-7871]. The high-resolution NMR structure of d(G(3)T(4)G(4))(2) G-quadruplex exhibits G-quadruplex core consisting of three stacked G-quartets. The two overhanging G3 and G11 residues are located at the opposite sides of the end G-quartets and are not involved in G-quartet formation. d(G(3)T(4)G(4))(2) G-quadruplex represents the first bimolecular G-quadruplex where end G-quartets are spanned by diagonal (T4-T7) as well as edge-type loops (T15-T18). Three of the G-rich strands are parallel while one is anti-parallel. The G12-G22 strand demonstrates a sharp reversal in strand direction between residues G19 and G20 that is accommodated with the leap over the middle G-quartet. The reversal in strand direction is achieved without any extra intervening residues. Here we furthermore examined the influence of different monovalent cations on the folding of d(G(3)T(4)G(4)). The resolved imino and aromatic proton resonances as well as (sequential) NOE connectivity patterns showed only minor differences in key intra- and interquartet NOE intensities in the presence of K(+), Na(+) and NH(4)(+) ions, which were consistent with subtle structural differences while retaining the same folding topology of d(G(3)T(4)G(4))(2) G-quadruplex.

15NH4+ ion movement inside d(G4T4G4)2 G-quadruplex is accelerated in the presence of smaller Na+ ions.

2D NMR studies demonstrate that the residence lifetime of 15NH4+ ions within the bimolecular G-quadruplex adopted by d(G4T4G4) is reduced from 270 ms in the presence of ammonium ions alone to 36 ms in the presence of Na+ ions.

Small change in a G-rich sequence, a dramatic change in topology: new dimeric G-quadruplex folding motif with unique loop orientations.

NMR study has shown that DNA oligonucleotide d(G(3)T(4)G(4)) adopts an asymmetric bimolecular G-quadruplex structure in solution. The structure of d(G(3)T(4)G(4))(2) is composed of three G-quartets, overhanging G11 residue and G3, which is part of the loop. Unique structural feature of d(G(3)T(4)G(4))(2) fold is the orientation of the two loops. Thymidine residues T4-T7 form a diagonal loop, whereas T15-T18 form an edge type loop. The G-quadruplex core of d(G(3)T(4)G(4))(2) consists of two stacked G-quartets with syn-anti-anti-anti alternation of dG residues and one G-quartet with syn-syn-anti-anti alternation. Another unusual structural feature of d(G(3)T(4)G(4))(2) is a leap between G19 and G20 over the middle G-quartet and chain reversal between G19 and G20 residues. The presence of one antiparallel and three parallel strands reveals the hitherto unknown G-quadruplex folding motif consisting of antiparallel/parallel strands and diagonal as well as edge type loops. Further examination of the influence of different monovalent cations on the folding of d(G(3)T(4)G(4)) showed that it forms a bimolecular G-quadruplex in the presence of K+, Na+, and NH4+ ions with the same general fold.

The solution structure of d(G(4)T(4)G(3))(2): a bimolecular G-quadruplex with a novel fold.

The G-rich 11-mer oligonucleotide d(G(4)T(4)G(3)) forms a bimolecular G-quadruplex in the presence of sodium ions with a topology that is distinct from the folds of the closely related and well-characterized sequences d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)). The solution structure of d(G(4)T(4)G(3))(2) has been determined using a combination of NMR spectroscopy and restrained molecular dynamics calculations. d(G(4)T(4)G(3))(2) forms an asymmetric dimeric fold-back structure consisting of three stacked G-quartets. The two T(4) loops that span diagonally across the outer faces of the G-quartets assume different conformations. The glycosidic torsion angle conformations of the guanine bases are 5'-syn-anti-syn-anti-(T(4) loop)-anti-syn-anti in one strand and 5'-syn-anti-syn-anti-(T(4) loop)-syn-anti-syn in the other strand. The guanine bases of the two outer G-quartets exhibit a clockwise donor-acceptor hydrogen-bonding directionality, while those of the middle G-quartet exhibit the anti-clockwise directionality. The topology of this G-quadruplex, like other bimolecular fold-back structures with diagonal loops, places each strand of the G-quartet region next to a neighboring parallel and an anti-parallel strand. The two guanine residues not involved in G-quartet formation, G4 and G12 (i.e. the fourth guanine base of one strand and the first guanine base of the other strand), adopt distinct conformations. G4 is stacked on top of an adjacent G-quartet, and this base-stacking continues along with the bases of the loop residues T5 and T6. G12 is orientated away from the core of G-quartets; stacked on the T7 base and apparently involved in hydrogen-bonding interactions with the phosphodiester group of this same residue. The cation-dependent folding of the d(G(4)T(4)G(3))(2) quadruplex structure is distinct from that observed for similar sequences. While both d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)) form bimolecular, diagonally looped G-quadruplex structures in the presence of Na(+), K(+) and NH(4)(+), we have observed this folding to be favored for d(G(4)T(4)G(3)) in the presence of Na(+), but not in the presence of K(+) or NH(4)(+). The structure of d(G(4)T(4)G(3))(2) exhibits a "slipped-loop" element that is similar to what has been proposed for structural intermediates in the folding pathway of some G-quadruplexes, and therefore provides support for the feasibility of these proposed transient structures in G-quadruplex formation.