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Resistance mechanisms against whole classes of antibiotics are currently developing faster than research generates new structurally different biologically active agents. The demand for new antimicrobial drugs has not been matched by the speed of discovery. The interface between sigma and core of bacterial RNA polymerase offers an attractive target for drug discovery, and we have previously described the development of a very robust high-throughput assay for this target based on luminescence resonance energy transfer. Here we describe a semiautomated screen of a commercially available library (Chembridge, San Diego, CA) that led to the identification of four small molecules, two of which have activity in preventing in vitro transcription and growth of Escherichia coli.

The "B-finger" of transcription factor IIB (TFIIB) is highly conserved and believed to play a role in the initiation process. We performed alanine substitutions across the B-finger of human TFIIB, made change-of-charge mutations in selected residues, and substituted the B-finger sequence from other organisms. Mutant proteins were examined in two minimal promoter systems (containing only RNA polymerase II, TATAbinding protein, and TFIIB) and in a complex system, using TFIIB-immunodepleted HeLa cell nuclear extract (NE). Mutations in conserved residues located on the sides of the B-finger had the greatest effect on activity in both minimal promoter systems, with mutations in residues Glu-51 and Arg-66 eliminating activity. The double change-of-charge mutant (E51R: R66E) did not show activity in either minimal promoter system. Mutations in the nonconserved residues at the tip of the B-finger did not significantly affect activity. However, all of the mutations in the B-finger showed at least 25% activity in the HeLa cell NE. Chimeric proteins, containing B-finger sequences from species with conserved residues on the side of the B-finger, showed wild-type activity in a minimal promoter system and in the HeLa cell NE. However, chimeric proteins whose sequence showed divergence on the sides of the B-finger had reduced activity. Transcription factor IIF (TFIIF) partially restored activity of the inactive mutants in the minimal promoter system, suggesting that TFIIF in HeLa cell NE helps to rescue the inactive mutations by interacting with either the B-finger or another component of the initiation complex that is influenced by the B-finger.The RNA polymerase II (RNAP II) 2 initiation complex is extremely complicated and, thus, very difficult to meaningfully dissect. Genes that are transcribed into mRNA by RNAP II generally require five general transcription factors (TFs), designated TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in Refs. 1-5). However, arguments have been made to include the "elongation" factor TFIIS (6) and the mediator complex (7) in the list of "general" transcription factors. RNAP II and most of the TFs are multimeric protein complexes. If all proteins contained in the yeast (Saccharomyces cerevisiae) RNAP II initiation complex were considered, the count of ϳ60 polypeptides would have a mass of ϳ3 MDa (8). The high resolution crystal structure of yeast RNAP II has provided invaluable insight into the topology of the RNAP II transcription initiation complex (9). Furthermore, the crystal structures of bacterial RNAP (10, 11) and RNAP from an archaeal organism (12) establish that RNAP from all three domains of life (Bacteria, Archaea, and Eukarya) show high conservation of overall structure. However, many mechanistic details of the transcription process have yet to be determined.TFIIB functions as a single polypeptide with a two-domain structure. The C-terminal domain of TFIIB (cTFIIB) contains an imperfect direct repeat motif. The co-crystal structure of cTFIIB with the DNA-bound TATA-binding protein ...

The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of σ70 with theβ' coiled-coil fragment (amino acids 100–309). Here we describe an LRET binding assay used to monitor the interaction of E. coli σ70 and σ32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of σ70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the σ32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of σ32 with core RNAP is stronger than σ70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E.coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function.

Synthetic derivatives of the natural product antibiotic novobiocin were synthesized in order to improve their physiochemical properties. A Mannich reaction was used to introduce new side chains at a solvent-exposed position of the molecule, and a diverse panel of functional groups was evaluated at this position. Novobiocin and the new derivatives were tested for their binding to gyrase B and their antibacterial activities against S. aureus, M. tuberculosis, F. tularensis and E. coli. While the new derivatives still bound the gyrase B protein potently (0.07 – 1.8 μM IC50), they had significantly less antibacterial activity. Two compounds were identified with increased antibacterial activity against M. tuberculosis, with a minimum inhibitory concentration of 2.5 μg/ml.

DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A2B2 heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin–Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-μL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration–approved compounds. The assay performed with an average Z′ factor of 0.80 and was able to identify GyrB inhibitors from a screening library.

BackgroundDisialoganglioside 2 (GD2) is expressed on neuroblastomas as well as melanomas, small cell lung cancers, and sarcomas. Anti-GD2 mAb (Dinutuximab) can be used to treat these cancers and is part of the standard care for neuroblastoma. While GD2 is expressed minimally on most normal tissues, it is expressed on some nerve cells, and anti-GD2 treatment can cause neuropathic pain. A separate tumor-antigen, B7H3, is overexpressed on multiple tumor types, including those listed above, with minimal expression on most normal cells and no expression on nerve cells. We developed a bispecific SNIPER antibody, INV721, to simultaneously target these 2 tumor antigens, with one arm specific to GD2 and the other arm to B7H3. The individual Fab arms targeting GD2 and B7H3 are each low to moderate affinity, such that INV721 will only bind with high affinity when both arms bind to their antigens on the same cell, resulting in high-specificity of the SNIPER to tumor cells.MethodsINV721 binding to GD2/B7H3-expressing tumors was confirmed by flow cytometry, as well as in tumor-bearing mice injected with 89Zr-labeled to monitor in vivo biodistribution via positron emission tomography imaging. Antibody-dependent cellular cytotoxicity (ADCC) testing of INV721 was performed on human neuroblastoma and melanoma cell lines with an Incucyte spheroid-killing-assay. In vivo efficacy studies were carried out in mice bearing GD2/B7H3-expressing melanoma tumors to test our in situ vaccine (ISV) regimen, which included testing combinations of external beam radiation therapy (RT, 12Gy) ± INV721 (40 ug/dose) ± IL2 (75K U/dose).ResultsINV721 showed binding by flow cytometry to tumors that express both GD2 and B7H3 but minimal binding to cells that don’t express both antigens. 89Zr-INV721 showed elevated and persistent accumulation in the tumor with minimal uptake in normal tissues. Incucyte spheroid-killing assays revealed that INV721 was capable of ADCC. The ISV combination of RT+INV721+IL2 was capable of curing mice bearing ~57 mm3 melanoma tumors (12/12 mice tumor free), with >70% of these mice exhibiting long-term immune memory.ConclusionsINV721 binds to cells that express both GD2 and B7H3, and these preliminary studies show that INV721 is effective in our ISV regimen at curing mice bearing tumors that express these antigens. We are continuing our efforts to determine if INV721 is associated with less pain than Dinutuximab. The goal of this SNIPER-antibody is to enhance the tumor-specific delivery of therapeutic mAbs, which may decrease toxicity and improve efficacy for cancers expressing both GD2 and B7H3.

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