Novel inhibitors that target bacterial virulence identified via HTS against intra-macrophage survival of Shigella flexneri.

Shigella flexneri is a facultative intracellular pathogen that causes shigellosis, a human diarrheal disease characterized by the destruction of the colonic epithelium. Novel antimicrobial compounds to treat infections are urgently needed due to the proliferation of bacterial antibiotic resistance and lack of new effective antimicrobials in the market. Our approach to find compounds that block the Shigella virulence pathway has three potential advantages: (i) resistance development should be minimized due to the lack of growth selection pressure, (ii) no resistance due to environmental antibiotic exposure should be developed since the virulence pathways are not activated outside of host infection, and (iii) the normal intestinal microbiota, which do not have the targeted virulence pathways, should be unharmed. We chose to utilize two phenotypic assays, inhibition of Shigella survival in macrophages and Shigella growth inhibition (minimum inhibitory concentration), to interrogate the 1.7 M compound screening collection subset of the GlaxoSmithKline drug discovery chemical library. A number of secondary assays on the hit compounds resulting from the primary screens were conducted, which, in combination with chemical, structural, and physical property analyses, narrowed the final hit list to 44 promising compounds for further drug discovery efforts. The rapid development of antibiotic resistance is a critical problem that has the potential of returning the world to a "pre-antibiotic" type of environment, where millions of people will die from previously treatable infections. One relatively newer approach to minimize the selection pressures for the development of resistance is to target virulence pathways. This is anticipated to eliminate any resistance selection pressure in environmental exposure to virulence-targeted antibiotics and will have the added benefit of not affecting the non-virulent microbiome. This paper describes the development and application of a simple, reproducible, and sensitive assay to interrogate an extensive chemical library in high-throughput screening format for activity against the survival of Shigella flexneri 2457T-nl in THP-1 macrophages. The ability to screen very large numbers of compounds in a reasonable time frame (~1.7 M compounds in ~8 months) distinguishes this assay as a powerful tool in further exploring new compounds with intracellular effect on S. flexneri or other pathogens with similar pathways of pathogenesis. The assay utilizes a luciferase reporter which is extremely rapid, simple, relatively inexpensive, and sensitive and possesses a broad linear range. The assay also utilized THP-1 cells that resemble primary monocytes and macrophages in morphology and differentiation properties. THP-1 cells have advantages over human primary monocytes or macrophages because they are highly plastic and their homogeneous genetic background minimizes the degree of variability in the cell phenotype (1). The intracellular and virulence-targeted selectivity of our methodology, determined via secondary screening, is an enormous advantage. Our main interest focuses on hits that are targeting virulence, and the most promising compounds with adequate physicochemical and drug metabolism and pharmacokinetic (DMPK) properties will be progressed to a suitable in vivo shigellosis model to evaluate the therapeutic potential of this approach. Additionally, compounds that act via a host-directed mechanism could be a promising source for further research given that it would allow a whole new, specific, and controlled approach to the treatment of diseases caused by some pathogenic bacteria.

Elucidation of the DNA-Binding Activity of VirF from Shigella flexneri for the icsA and rnaG Promoters and Characterization of the N-Terminal Domain To Identify Residues Crucial for Dimerization.

A novel approach to treat the highly virulent and infectious enteric pathogen Shigella flexneri, with the potential for reduced resistance development, is to target virulence pathways. One promising such target is the AraC-family transcription factor VirF, which activates downstream virulence factors. VirF harbors a conserved C-terminal DNA-binding domain (DBD) and an N-terminal dimerization domain (NTD). Previously, we studied the wild type (WT) and seven alanine DBD mutants of VirF binding to the virB promoter (N. J. Ragazzone, G. T. Dow, and A. Garcia, J Bacteriol 204:e00143-22, 2022, https://doi.org/10.1128/jb.00143-22). Here, we report studies of VirF binding to the icsA and rnaG promoters. Gel shift assays (electrophoretic mobility shift assays [EMSAs]) of WT VirF binding to these promoters revealed multiple bands at higher apparent molecular weights, indicating the likelihood of VirF dimerization when bound to DNA. For three of the mutants, we observed consistent effects on binding to the three promoters. For the four other mutants, we observed differential effects on promoter binding. Results of a cell-based, LexA monohybrid ß-galactosidase reporter assay [D. A. Daines, M. Granger-Schnarr, M. Dimitrova, and R. P. Silver, Methods Enzymol 358:153-161, 2002, https://doi.org/10.1016/s0076-6879(02)58087-3] indicated that WT VirF dimerizes in vivo and that alanine mutations to Y132, L137, and L147 significantly reduced dimerization. However, these mutations negatively impacted protein stability. We did purify enough of the Y132A mutant to determine that it binds in vitro to the virB and rnaG promoters, albeit with weaker affinities. Full-length VirF model structures, generated with I-TASSER, predict that these three amino acids are in a "dimerization" helix in the NTD, consistent with our results. IMPORTANCE Antimicrobial-resistant (AMR) infections continue to rise dramatically, and the lack of new approved antibiotics is not ameliorating this crisis. A promising route to reduce AMR is by targeting virulence. The Shigella flexneri virulence pathway is a valuable source for potential therapeutic targets useful to treat this infection. VirF, an AraC-family virulence transcription factor, is responsible for activating necessary downstream virulence genes that allow the bacteria to invade and spread within the human colon. Previous studies have identified how VirF interacts with the virB promoter and have even developed a lead DNA-binding inhibitor, but not much is known about VirF dimerization or binding to the icsA and rnaG promoters. Fully characterizing VirF can be a valuable resource for inhibitor discovery/design.

Elucidation of Key Interactions between VirF and the virB Promoter in Shigella flexneri Using E. coli MarA- and GadX-Based Homology Models and In Vitro Analysis of the DNA-Binding Domains of VirF and MarA.

Infection with Shigella, the organism responsible for the diarrheal disease shigellosis, leads to approximately 200,000 deaths globally annually. Virulence of this pathogen is primarily controlled by the DNA-binding transcriptional activator VirF. This AraC family protein activates transcription of two major virulence genes, virB and icsA, which lead to the pathogen's ability to invade and spread within colonic epithelial cells. While several AraC proteins have been studied, few studies of VirF's binding to its DNA promoters have been reported, and VirF's three-dimensional structure remains unsolved. Here, we used structures of two E. coli VirF homologs, GadX and MarA-marRAB, to generate homology models of the VirF DNA-binding domain in free and DNA-bound conformations. We conducted alanine scanning mutagenesis on seven residues within MarA that make base-specific interactions with its promoter, marRAB, and the corresponding residues within VirF (identified by sequence and structural homologies). In vitro DNA-binding assays studying both wild-type and mutant MarA and VirF proteins identified residues important for binding to the marRAB and virB promoters, respectively. Comparison of the effects of these DNA-binding domain mutants validated our MarA-based homology model, allowing us to identify crucial interactions between VirF and the virB promoter. Proteins with mutations to helix 3 within both MarA(W42A, R46A) and MalE-VirF(R192A, K193A) exhibited significant reductions in DNA binding, while the effects of mutations in helix 6 varied. This suggests the shared importance of helix 3 in the binding to these promoters, while helix 6 is transcription factor specific. These results can inform further development of virulence-targeting inhibitors as an alternative to traditional antimicrobial drug design. IMPORTANCE Globally, infection with Shigella flexneri is a leading cause of bacterial dysentery, particularly affecting children under the age of 5 years. The virulence of this pathogen makes it highly infectious, allowing it to spread easily within areas lacking proper sanitation or access to clean drinking water. VirF is a DNA-binding transcription factor that activates S. flexneri virulence once the bacteria infect the human colon. Development of drugs that target VirF's DNA-binding activity can be an effective treatment to combat shigellosis as an alternative or addition to traditional antibiotics. Due to the lack of structural data, analysis of VirF's DNA-binding activity is critical to the development of potent VirF inhibitors.

Optimization of Benzoxazinorifamycins to Minimize hPXR Activation for the Treatment of Tuberculosis and HIV Coinfection.

Tuberculosis (TB) is one of the most significant world health problems, responsible for 1.5 M deaths in 2020, and yet, current treatments rely largely on 40 year old paradigms. Although the rifamycins (RIFs), best exemplified by the drug rifampin (RMP), represent a well-studied and therapeutically effective chemotype that targets the bacterial RNA polymerase (RNAP), these agents still suffer from serious drawbacks including the following: 3-9 month treatment times; cytochrome P450 (Cyp450) induction [particularly problematic for human immunodeficiency virus-Mycobacterium tuberculosis (MTB) co-infection]; and the existence of RIF-resistant (RIFR) MTB strains. We have utilized a structure-based drug design approach to synthesize and test 15 benzoxazinorifamycins (bxRIFs), congeners of the clinical candidate rifalazil, to minimize human pregnane X receptor (hPXR) activation while improving potency against MTB. We have determined the compounds' activation of the hPXR [responsible for inducing Cyp450 3A4 (CYP3A4)]. Compound IC50s have been determined against the wild-type and the most prevalent RIFR (ß-S450L) mutant MTB RNAPs. We have also determined their bactericidal activity against "normal" replicating MTB and a model for non-replicating, persister MTB. We have identified a minimal substitution and have probed larger substitutions that exhibit negligible hPXR activation (1.2-fold over the dimethyl sulfoxide control), many of which are 5- to 10-fold more potent against RNAPs and MTB than RMP. Importantly, we have analogues that are essentially equipotent against replicating MTB and non-replicating persister MTB, a property that is correlated with faster kill rates and may lead to shorter treatment durations. This work provides a proof of principle that the ansamycin core remains an attractive and effective scaffold for novel and dramatically improved RIFs.

Optimization of Benzoxazinorifamycins to Improve Mycobacterium tuberculosis RNA Polymerase Inhibition and Treatment of Tuberculosis.

Rifampin (RMP), a very potent inhibitor of the Mycobacterium tuberculosis (MTB) RNA polymerase (RNAP), remains a keystone in the treatment of tuberculosis since its introduction in 1965. However, rifamycins suffer from serious drawbacks, including 3- to 9-month treatment times, Cyp450 induction (particularly problematic for HIV-MTB coinfection), and resistant mutations within RNAP that yield RIF-resistant (RIFR) MTB strains. There is a clear and pressing need for improved TB therapies. We have utilized a structure-based drug design approach to synthesize and test novel benzoxazinorifamycins (bxRIF), congeners of the clinical candidate rifalazil. Our goal is to gain binding interactions that will compensate for the loss of RIF-binding affinity to the (RIFR) MTB RNAP and couple those with substitutions that we have previously found that essentially eliminate Cyp450 induction. Herein, we report a systematic exploration of 42 substituted bxRIFs that have yielded an analogue (27a) that has an excellent in vitro activity (MTB RNAP inhibition, MIC, MBC), enhanced (∼30-fold > RMP) activity against RIFR MTB RNAP, negligible hPXR activation, good mouse pharmacokinetics, and excellent activity with no observable adverse effects in an acute mouse TB model. In a time-kill study, 27a has a 7 day MBC that is ∼10-fold more potent than RMP. These results suggest that 27a may exhibit a faster kill rate than RMP, which could possibly reduce the clinical treatment time. Our synthetic protocol enabled the synthesis of ∼2 g of 27a at >95% purity in 3 months, demonstrating the feasibility of scale-up synthesis of bxRIFs for preclinical and clinical studies.

Metal-HisTag coordination for remote loading of very small quantities of biomacromolecules into PLGA microspheres.

Challenges to discovery and preclinical development of long-acting release systems for protein therapeutics include protein instability, use of organic solvents during encapsulation, specialized equipment and personnel, and high costs of proteins. We sought to overcome these issues by combining remote-loading self-healing encapsulation with binding HisTag protein to transition metal ions. Porous, drug-free self-healing microspheres of copolymers of lactic and glycolic acids with high molecular weight dextran sulfate and immobilized divalent transition metal (M2+) ions were placed in the presence of proteins with or without HisTags to bind the protein in the pores of the polymer before healing the surface pores with modest temperature. Using human serum albumin, insulin-like growth factor 1, and granulocyte-macrophage colony-stimulating factor (GM-CSF), encapsulated efficiencies of immunoreactive protein relative to nonencapsulation protein solutions increased from ~41%, ~23%, and ~9%, respectively, without Zn2+ and HisTags to ~100%, ~83%, and ~75% with Zn2+ and HisTags. These three proteins were continuously released in immunoreactive form over seven to ten weeks to 73%-100% complete release, and GM-CSF showed bioactivity >95% relative to immunoreactive protein throughout the release interval. Increased encapsulation efficiencies were also found with other divalent transition metals ions (Co2+, Cu2+, Ni2+, and Zn2+), but not with Ca2+. Ethylenediaminetetraacetic acid was found to interfere with this process, reverting encapsulation efficiency back to Zn2+-free levels. These results indicate that M2+-immobilized self-healing microspheres can be prepared for simple and efficient encapsulation by simple mixing in aqueous solutions. These formulations provide slow and continuous release of immunoreactive proteins of diverse types by using a amount of protein (e.g., <10 µg), which may be highly useful in the discovery and early preclinical development phase of new protein active pharmaceutical ingredients, allowing for improved translation to further development of potent proteins for local delivery.

High-throughput screening to discover inhibitors of the CarD·RNA polymerase protein-protein interaction in Mycobacterium tuberculosis.

Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) accounts for 3.7% of new cases of TB annually worldwide and is a major threat to global public health. Due to the prevalence of the MDR-TB and extensively drug resistant tuberculosis (XDR-TB) cases, there is an urgent need for new drugs with novel mechanisms of action. CarD, a global transcription regulator in MTB, binds RNAP and activates transcription by stabilizing the transcription initiation open-promoter complex (RPo). CarD is required for MTB viability and it has highly conserved homologues in many eubacteria. A fluorescence polarization (FP) assay which monitors the association of MTB RNAP, native rRNA promoter DNA and CarD has been developed. Overall, our objective is to identify and characterize small molecule inhibitors which block the CarD/RNAP interaction and to understand the mechanisms by which CarD interacts with the molecules. We expect that the development of a new and improved anti-TB compound with a novel mechanism of action will relieve the burden of resistance. This CarD FP assay is amenable to HTS and is an enabling tool for future novel therapeutic discovery.

Source of the Fitness Defect in Rifamycin-Resistant Mycobacterium tuberculosis RNA Polymerase and the Mechanism of Compensation by Mutations in the ß' Subunit.

Mycobacterium tuberculosis is a critical threat to human health due to the increased prevalence of rifampin resistance (RMPr). Fitness defects have been observed in RMPr mutants with amino acid substitutions in the ß subunit of RNA polymerase (RNAP). In clinical isolates, this fitness defect can be ameliorated by the presence of secondary mutations in the double-psi ß-barrel (DPBB) domain of the ß' subunit of RNAP. To identify factors contributing to the fitness defects observed in vivo, several in vitro RNA transcription assays were utilized to probe initiation, elongation, termination, and 3'-RNA hydrolysis with the wild-type and RMPrM. tuberculosis RNAPs. We found that the less prevalent RMPr mutants exhibit significantly poorer termination efficiencies relative to the wild type, an important factor for proper gene expression. We also found that several mechanistic aspects of transcription of the RMPr mutant RNAPs are impacted relative to the wild type. For the clinically most prevalent mutant, the ßS450L mutant, these defects are mitigated by the presence of secondary/compensatory mutations in the DPBB domain of the ß' subunit.

Structural basis for rifamycin resistance of bacterial RNA polymerase by the three most clinically important RpoB mutations found in Mycobacterium tuberculosis.

Since 1967, Rifampin (RMP, a Rifamycin) has been used as a first line antibiotic treatment for tuberculosis (TB), and it remains the cornerstone of current short-term TB treatment. Increased occurrence of Rifamycin-resistant (RIFR ) TB, ∼41% of which results from the RpoB S531L mutation in RNA polymerase (RNAP), has become a growing problem worldwide. In this study, we determined the X-ray crystal structures of the Escherichia coli RNAPs containing the most clinically important S531L mutation and two other frequently observed RIFR mutants, RpoB D516V and RpoB H526Y. The structures reveal that the S531L mutation imparts subtle if any structural or functional impact on RNAP in the absence of RIF. However, upon RMP binding, the S531L mutant exhibits a disordering of the RIF binding interface, which effectively reduces the RMP affinity. In contrast, the H526Y mutation reshapes the RIF binding pocket, generating significant steric conflicts that essentially prevent any RIF binding. While the D516V mutant does not exhibit any such gross structural changes, certainly the electrostatic surface of the RIF binding pocket is dramatically changed, likely resulting in the decreased affinity for RIFs. Analysis of interactions of RMP with three common RIFR mutant RNAPs suggests that modifications to RMP may recover its efficacy against RIFR TB.

Novel Chemical Scaffolds for Inhibition of Rifamycin-Resistant RNA Polymerase Discovered from High-Throughput Screening.

Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (RifR) strains of Mycobacterium tuberculosis (MTB). To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that uses malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant RifR RNAP (ßS531L) containing a mutation (ß'V408G) that compensates for the fitness defect of this RifR mutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of wild-type and RifR E. coli and MTB RNAPs. Four compounds identified from the screen are active against MTB in culture at concentrations below 200 µM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan-assay interference.

Academic Freedom Should Be Redefined: Point and Counterpoint.

As part of the 2014-15 Academic Leadership Fellows Program, the cohort teams presented debates on topics relevant to academic pharmacy at a public forum during the 2015 American Association of Colleges of Pharmacy Interim Meeting. The topic of one of the debates was "Academic Freedom Should Be Redefined." The "point" of the debate focused on important issues such as the fundamental definition of academic freedom as it was written in the 1940 American Association of University Professors' Statement and the need for redefinition as a consequence of many misunderstandings and misinterpretations that have arisen over time. The "counterpoint" received the greatest support, and it asserted that redefinition is not necessary, but rather the need is to clearly articulate the intended meaning of academic freedom through education, discussion, and by not supporting inappropriate behaviors in the name of "academic freedom." Reinforced clarity and operational guidance from the academy and academic institutions may add further clarification and may be the best approach to address the concerns related to academic freedom.

Mechanism of Action and Initial, In Vitro SAR of an Inhibitor of the Shigella flexneri Virulence Regulator VirF.

Shigella spp. are among the main causative agents of acute diarrheal illness and claim more than 1 million lives per year worldwide. There are multiple bacterial genes that control the pathogenesis of Shigella, but the virF gene may be the most important. This gene, located on the primary pathogenicity island of Shigella, encodes VirF, an AraC-family transcriptional activator that is responsible for initiating the pathogenesis cycle in Shigella. We have previously shown that it is possible to attenuate the virulence of Shigella flexneri via small molecule inhibition of VirF. In this study, we probed the mechanism of action of our small molecule inhibitors of VirF. To enable these studies, we have developed a homologous and efficient expression and purification system for VirF and have optimized two different in vitro VirF-DNA binding assays. We have determined that one of our HTS hit compounds inhibits VirF binding to DNA with a calculated Ki similar to the effective doses seen in our transcriptional activation and virulence screens. This is consistent with inhibition of DNA binding as the mechanism of action of this hit compound. We have also screened 15 commercially sourced analogs of this compound and deduced an initial SAR from the approximately 100-fold range in activities. Our four other HTS hit compounds do not inhibit DNA binding and yet they do block VirF activity. This suggests that multiple agents with different molecular mechanisms of inhibition of VirF could be developed. Pursuing hits with different mechanisms of action could be a powerful approach to enhance activity and to circumvent resistance that could develop to any one of these agents.

Potential novel antibiotics from HTS targeting the virulence-regulating transcription factor, VirF, from Shigella flexneri.

VirF is an AraC-type transcriptional regulator responsible for activating the transcription of virulence genes required for the intracellular invasion and cell-to-cell spread of Shigella flexneri. Gene disruption studies have validated VirF as a potential target for an anti-virulence therapy to treat shigellosis by determining that VirF is necessary for virulence, but not required for bacterial viability. Using a bacteria-based, ß-galactosidase reporter assay we completed a high-throughput screening (HTS) campaign monitoring VirF activity in the presence of over 140,000 small molecules. From our screening campaign, we identified five lead compounds to pursue in tissue culture-based invasion and cell-to-cell spread assays, and toxicity screens. Our observations of activity in these models for infection have validated our approach of targeting virulence regulation and have allowed us to identify a promising chemical scaffold from our HTS for hit-to-lead development. Interestingly, differential effects on invasion versus cell-to-cell spread suggest that the compounds' efficacies may depend, in part, on the specific promoter that VirF is recognizing.

X-ray crystal structures of the Escherichia coli RNA polymerase in complex with benzoxazinorifamycins.

Rifampin, a semisynthetic rifamycin, is the cornerstone of current tuberculosis treatment. Among many semisynthetic rifamycins, benzoxazinorifamycins have great potential for TB treatment due to their superior affinity for wild-type and rifampin-resistant Mycobacterium tuberculosis RNA polymerases and their reduced hepatic Cyp450 induction activity. In this study, we have determined the crystal structures of the Escherichia coli RNA polymerase complexes with two benzoxazinorifamycins. The ansa-naphthalene moieties of the benzoxazinorifamycins bind in a deep pocket of the ß subunit, blocking the path of the RNA transcript. The C3'-tail of benzoxazinorifamycin fits a cavity between the ß subunit and σ factor. We propose that in addition to blocking RNA exit, the benzoxazinorifamycin C3'-tail changes the σ region 3.2 loop position, which influences the template DNA at the active site, thereby reducing the efficiency of transcription initiation. This study supports expansion of structure-activity relationships of benzoxazinorifamycins inhibition of RNA polymerase toward uncovering superior analogues with development potential.

Investigating the prevalence of queuine in Escherichia coli RNA via incorporation of the tritium-labeled precursor, preQ(1).

There are over 100 modified bases that occur in RNA with the majority found in transfer RNA. It has been widely believed that the queuine modification is limited to four transfer RNA species in vivo. However, given the vast amount of the human genome (60-70%) that is transcribed into non-coding RNA (Mattick [10]), probing the presence of modified bases in these RNAs is of fundamental importance. The mechanism of incorporation of queuine, via transglycosylation, makes this uniquely poised to probe base modification in RNA. Results of incubations of Escherichia coli cell cultures with [(3)H] preQ(1) (a queuine precursor in eubacteria) clearly demonstrate preQ(1) incorporation into a number of RNA species of various sizes larger than transfer RNA. Specifically, significant levels of preQ(1) incorporation into ribosomal RNA are observed. The modification of other large RNAs was also observed. These results confirm that non-coding RNAs contain modified bases and lead to the supposition that these modifications are necessary to control non-coding RNA structure and function as has been shown for transfer RNA.

Cell permeable cocaine esterases constructed by chemical conjugation and genetic recombination.

Cocaine esterase (CocE) is the most efficient cocaine-metabolizing enzyme tested in vivo to date, displaying a rapid clearance of cocaine and a robust protection against cocaine's toxicity. Two potential obstacles to the clinical application of CocE, however, lie in its proteolytic degradation and induced immune response. To minimize these potential obstacles, we attempted nondisruptive cell encapsulation by creating a cell permeable form of CocE, which was achieved by covalently linking a thermally stable CocE mutant (dmCocE) with cell penetrating peptides (CPPs). Two types of CPPs, Tat and the low molecular weight protamine (LMWP), were used in this study. Two types of disulfide-bridged chemical conjugates, Tat-S-S-dmCocE and LMWP-S-S-dmCocE, were synthesized and then purified by heparin affinity chromatography. In addition, four recombinant CPP-dmCocE fusion proteins, Tat-N-dmCocE, LMWP-N-dmCocE, dmCocE-C-Tat, and dmCocE-C-LMWP, were constructed, expressed in Escherichia coli, and purified as soluble proteins. Among these six CPP-dmCocE variants, LMWP-S-S-dmCocE showed the highest cocaine-hydrolyzing activity, and dmCocE-C-Tat had the highest production yield. To evaluate their cellular uptake behavior, a covalently linked fluorophore (FITC) was utilized to visualize the cellular uptake of all six CPP-dmCocE variants in living HeLa cells. All the six variants exhibited cellular uptake, but their intracellular distribution phenotypes differed. While the chemical conjugates showed primarily cytoplasmic distribution, which was likely due to the reduction of the disulfide linkage between CPP and dmCocE, all the other four recombinant fusion proteins displayed both nuclear and cytoplasmic localization, with dmCocE-C-CPP exhibiting higher cytoplasmic distribution during cellular uptake. Based on a balanced consideration of essentials for clinical application, including parameters such as high cocaine-hydrolyzing efficiency, large production yield, major cytoplasmic distribution, etc., the dmCocE-C-Tat fusion protein seems to be the best candidate from this investigation. Further in vivo studies of the cell-encapsulated dmCocE-C-Tat in hydrolyzing cocaine and alleviating immunogenicity and proteolytic degradation in established, clinically relevant mouse models are currently underway in our laboratories. Findings from this research are not only useful for developing other new CPP-CocE constructs but also valuable for establishing a nondisruptive cell-encapsulation technology for other protein therapeutics that are known to be immunogenic for direct clinical application.

Structure-based design of novel benzoxazinorifamycins with potent binding affinity to wild-type and rifampin-resistant mutant Mycobacterium tuberculosis RNA polymerases.

By utilization of three-dimensional structure information of rifamycins bound to RNA polymerase (RNAP) and the human pregnane X receptor (hPXR), representative examples (2b-d) of a novel subclass of benzoxazinorifamycins have been synthesized. Relative to rifalazil (2a), these analogues generally display superior affinity toward wild-type and Rif-resistant mutants of the Mycobacterium tuberculosis RNAP but lowered antitubercular activity in cell culture under both aerobic and anaerobic conditions. Lowered affinity toward hPXR for some of the analogues is also observed, suggesting a potential for reduced Cyp450 induction activity. Mouse and human microsomal studies of analogue 2b show it to have excellent metabolic stability. Mouse pharmacokinetics in plasma and lung show accumulation of 2b but with a half-life suggesting nonoptimal pharmacokinetics. These studies demonstrate proof of principle for this subclass of rifamycins and support further expansion of structure-activity relationships (SARs) toward uncovering analogues with development potential.

Synthesis and structure-activity relationships of novel substituted 8-amino, 8-thio, and 1,8-pyrazole congeners of antitubercular rifamycin S and rifampin.

A series of rifamycin S and rifampin analogues incorporating substituted 8-amino, 8-thio, and 1,8-pyrazole substituents has been synthesized. The compounds were made by activation of the C-8 phenol as a sulfonate ester, followed by displacement with selected nitrogen and sulfur nucleophiles. The analogues were screened in assays to quantify their antitubercular activity under both aerobic and anaerobic conditions, and for inhibition of wild-type Mycobacterium tuberculosis (MTB) RNAP and rifamycin-resistant MTB RNAP (S450L) via an in vitro rolling circle transcription assay. Additionally, the MIC(90) values were determined for these analogues against Escherichia coli strains. Although none of the analogues displayed superior enzymatic or microbiological activity to their parent scaffolds, the results are consistent with the Rif C-8 hydroxyl acting as a hydrogen bond acceptor with S450 and that Rif resistance in the S450L mutant is due to loss of this hydrogen bond. Representative analogues were also evaluated in the human pregnane X receptor (PXR) activation assay.

Differential heterocyclic substrate recognition by, and pteridine inhibition of E. coli and human tRNA-guanine transglycosylases.

tRNA-guanine transglycosylases (TGTs) are responsible for incorporating 7-deazaguanine-modified bases into certain tRNAs in eubacteria (preQ(1)), eukarya (queuine) and archaea (preQ(0)). In each kingdom, the specific modified base is different. We have found that the eubacterial and eukaryal TGTs have evolved to be quite specific for their cognate heterocyclic base and that Cys145 (Escherichia coli) is important in recognizing the amino methyl side chain of preQ(1) (Chen et al., Nuc. Acids Res. 39 (2011) 2834 [15]). A series of mutants of the E. coli TGT have been constructed to probe the role of three other active site amino acids in the differential recognition of heterocyclic substrates. These mutants have also been used to probe the differential inhibition of E. coli versus human TGTs by pteridines. The results indicate that mutation of these active site amino acids can "open up" the active site, allowing for the binding of competitive pteridine inhibitors. However, even the "best" of these mutants still does not recognize queuine at concentrations up to 50µM, suggesting that other changes are necessary to adapt the eubacterial TGT to incorporate queuine into RNA. The pteridine inhibition results are consistent with an earlier hypothesis that pteridines may regulate eukaryal TGT activity (Jacobson et al., Nuc. Acids Res. 9 (1981) 2351 [8]).