Impact of the structure-activity relationship of AHL analogues on quorum sensing in Gram-negative bacteria.

With the emergence of microbial resistance pathogens, recent research aims at studying new mechanisms of action of antibiotics. This review discusses the mechanisms and types of quorum sensing (QS) inhibitors in Gram negative bacteria. It illustrates all published data available in literature pertaining to novel compounds that showed activity against different targets in the quorum sensing pathways in Gram negative bacteria. A systemic overview has been conducted by searching PubMed, Medline, and the Cochrane Library and data extraction of all quorum sensing inhibitors with their mechanisms of action have been collected. This review will focus on signaling autoinducer AI-1 in Gram negative bacteria. The biological activity of the antagonists is mainly reported as IC50 (the concentration of an inhibitor where the response is reduced by half).

Recent progresses on synthesized LuxS inhibitors: A mini-review.

Design and synthesis of LuxS enzyme inhibitors otherwise known as S-ribosylhomocysteine analogues, to target quorum sensing in bacteria, has been considerably developed within the last decade. This review presents which molecules have been synthesized to target LuxS enzyme in other words inhibitors of S-ribosylhomocysteinase. It reports their tested biological activity as LuxS inhibitors when available. A systematic overview has been conducted by searching PubMed, Medline, and The Cochrane Library and data extraction of all synthesized S-ribosylhomocysteine analogues has been collected. This mini-review shows limited data to date on this area and should continue to be studied.

Synthesis of isomeric analogues of S-ribosylhomocysteine analogues with homocysteine unit attached to C2 of ribose.

LuxS (S-ribosylhomocysteinase; EC 4.4.1.21) is an enzyme that catalyzes the cleavage of the thioether linkage in the catalytic pathway of S-ribosylhomocysteine (SRH) which produces homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD). DPD is the precursor of the signaling molecules known as autoinducer 2 (AI-2) responsible for the bacterial quorum sensing (QS) identified as cell to cell communication. Inhibitors of LuxS should be able to interfere with its catalytic pathway thus preventing the formation of the autoinducer molecules. In this work, the synthesis of 2-deoxy-2-bromo-SRH analogues was attempted by the coupling of the corresponding 2-bromo-2-deoxypentafuranosyl sugars with the homocysteinate anion. The displacement of the bromide from C2 rather than the expected substitution of the mesylate group from C5 was observed leading to a novel isomeric analogue of SRH in which Hcy moiety is attached to a ribose ring via C2-sulfur bond.

S-Ribosylhomocysteine Analogues Modified at the Ribosyl C-4 Position.

4-C-Alkyl/aryl-S-ribosylhomocysteine (SRH) analogues were prepared by coupling of homocysteine with 4-substituted ribofuranose derivatives. The diastereoselective incorporation of the methyl substituent into the 4 position of the ribose ring was accomplished by addition of methylmagnesium bromide to the protected ribitol-4-ulose yielding the 4-C-methylribitol in 85% yield as single 4R diastereomer. The 4-C hexyl, octyl, vinyl, and aryl ribitols were prepared analogously. Chelation controlled addition of a carbanion to ketones from the (Si-face) was responsible for the observed stereochemical outcome. Oxidation of the primary alcohol of the 4-C ribitols with the catalytic amount of tetrapropylammonium perruthenate in the presence of N-methylmorpholine N-oxide produced 4-C-alkylribono-1,4-lactones in high yields. Mesylation of the latter compounds at the 5-hydroxyl position and treatment with a protected homocysteine thiolate afforded protected 4-C-alkyl/aryl-SRH analogues as the lactones. Reduction with lithium triethylborohydride and successive global deprotections with TFA afforded 4-C-alkyl/aryl SRH analogues. These analogues might impede the S-ribosylhomocysteinase(LuxS)-catalyzed reaction by preventing ß-elimination of a homocysteine molecule, and thus depleting the production of quorum sensing signaling molecule AI-2.

Potential of DPD ((S)-4,5-dihydroxy-2,3-pentanedione) Analogs in Microparticulate Formulation as Vaccine Adjuvants.

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is involved in bacterial communication. DPD is the precursor of signal molecule autoinducer-2 (AI-2) and has high potential to be used as a vaccine adjuvant. Vaccine adjuvants are compounds that enhance the stability and immunogenicity of vaccine antigens, modulate efficacy, and increase the immune response to a particular antigen. Previously, the microparticulate form of (S)-DPD was found to have an adjuvant effect with the gonorrhea vaccine. In this study, we evaluated the immunogenicity and adjuvanticity of several synthetic analogs of the (S)-DPD molecule, including ent-DPD((R)-4,5-dihydroxy-2,3-pentanedione), n-butyl-DPD ((S)-1,2-dihydroxy-3,4-octanedione), isobutyl-DPD ((S)-1,2-dihydroxy-6-methyl-3,4-heptanedione), n-hexyl-DPD ((S)-1,2-dihydroxy-3,4-decanedione), and phenyl-DPD ((S)-3,4-dihydroxy-1-phenyl-1,2-butanedione), in microparticulate formulations. The microparticulate formulations of all analogs of (S)-DPD were found to be noncytotoxic toward dendritic cells. Among these analogs, ent-DPD, n-butyl-DPD, and isobutyl-DPD were found to be immunogenic toward antigens and showed adjuvant efficacy with microparticulate gonorrhea vaccines. It was observed that n-hexyl-DPD and phenyl-DPD did not show any adjuvant effect. This study shows that synthetic analogs of (S)-DPD molecules are capable of eliciting adjuvant effects with vaccines. A future in vivo evaluation will further confirm that these analogs are promising vaccine adjuvants.

Evaluating the Release of Different Commercial Orally Modified Niacin Formulations In Vitro.

OBJECTIVES: To evaluate the release profile of different modified-release oral formulations of niacin, such as immediate-release (IR) powder and tablets, timed-release (TR) caplets, extended-release (ER) capsules, and controlled-release (CR) tablets, to assure their defined release pattern and correlate this release with their matrix polymers. SIGNIFICANCE: Niacin is used to manage hyperlipidemia by reducing cutaneous flushing and hepatotoxicity adverse events. The release profiles of different types of modified-release dosage forms depend on the types of coating materials (polymers) used in the matrix formation. Although different types of niacin formulations exist, none of the niacin dissolution profiles have been evaluated and compared in the literature. METHODS: Four commercial orally modified-release niacin brands were collected from a local CVS pharmacy retail store, in Miami, FL, USA. The in vitro release study was conducted in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) conditions. RESULTS: The results of the release patterns of four niacin-modified dosage forms (IR, ER, TR, and CR) were aligned with their release definitions. However, the CR dosage form did not follow an ideal release pattern. CONCLUSIONS: The release rate of niacin in vitro was pH dependent, which was confirmed by the similarity factor (f2) results. All the f2 comparison values were below 50 in both the SIF and SGF media, while all the comparisons were below the f2 values for all brands in the SIF media.

The Autoinducer N-Octanoyl-L-Homoserine Lactone (C8-HSL) as a Potential Adjuvant in Vaccine Formulations.

Autoinducers AI-1 and AI-2 play an important role in bacterial quorum sensing (QS), a form of chemical communication between bacteria. The autoinducer N-octanoyl-L-Homoserinehomoserine lactone (C8-HSL) serves as a major inter- and intraspecies communicator or 'signal', mainly for Gram-negative bacteria. C8-HSL is proposed to have immunogenic properties. The aim of this project is to evaluate C8-HSL as a potential vaccine adjuvant. For this purpose, a microparticulate formulation was developed. The C8-HSL microparticles (MPs) were formulated by a water/oil/water (W/O/W) double-emulsion solvent evaporation method using PLGA (poly (lactic-co-glycolic acid)) polymer. We tested C8-HSL MPs with two spray-dried bovine serum albumin (BSA)-encapsulated bacterial antigens: colonization factor antigen I (CFA/I) from Escherichia coli (E. coli.) and the inactive protective antigen (PA) from Bacillus anthracis (B. anthracis). We formulated and tested C8-HSL MP to determine its immunogenicity potential and its ability to serve as an adjuvant with particulate vaccine formulations. An in vitro immunogenicity assessment was performed using Griess's assay, which indirectly measures the nitric oxide radical (NOˑ) released by dendritic cells (DCs). The C8-HSL MP adjuvant was compared with FDA-approved adjuvants to determine its immunogenicity potential. C8-HSL MP was combined with particulate vaccines for measles, Zika and the marketed influenza vaccine. The cytotoxicity study showed that MPs were non-cytotoxic toward DCs. Griess's assay showed a comparable release of NOˑ from DCs when exposed to CFA and PA bacterial antigens. Nitric oxide radical (NOˑ) release was significantly higher when C8-HSL MPs were combined with particulate vaccines for measles and Zika. C8-HSL MPs showed immunostimulatory potential when combined with the influenza vaccine. The results showed that C8-HSL MPs were as immunogenic as FDA-approved adjuvants such as alum, MF59, and CpG. This proof-of-concept study showed that C8-HSL MP displayed adjuvant potential when combined with several particulate vaccines, indicating that C8-HSL MPs can increase the immunogenicity of both bacterial and viral vaccines.

Creation and implementation of a drug discovery and development game.

BACKGROUND AND PURPOSE: Gamification is a commonly employed active-learning technique to increase student engagement and learning. Few games teaching the drug discovery and development process exist. EDUCATIONAL ACTIVITY AND SETTING: A six hour component of the elective course Non-traditional Pharmacy Career Routes focused on drug development. Four of the six hours were devoted to a game designed to mimic the drug discovery and development process. The 17 enrolled students were split into smaller groups designated to represent large pharmaceutical, start-up, or generic companies. The number of resources each group began with varied depending on the type of company they were assigned. Students worked to develop and bring to market the most drugs and gain the most money. To reinforce the reflective and innovative learning process, students created "failure" cards before the game started that had reasons for failures during the drug development process. FINDINGS: Two questions about the drug discovery and development process were on a pre-/post-assessment. The first question was answered correctly by 12 of 16 students on the pre-assessment, while 15 of 17 students answered correctly on the post-assessment. The second question was answered correctly by 13 of 16 students on the pre-assessment and all students on the post-assessment. The students enjoyed playing the game and felt that it helped them to understand the drug development process. SUMMARY: A novel, role-play game that allows students to learn the drug discovery and development process has the potential to be implemented in similar courses.

Pre-clinical Impact of the Synergistic Mechanism of Daptomycin and Ceftaroline on Patients with Methicillin-resistant Staphylococcus aureus Bacteremia Infections.

BACKGROUND: Our study aims at assessing the pre-clinical impact of the synergistic mechanism of Daptomycin (DAP) and Ceftaroline (CFT) on patients with Methicillin-Resistant Staphylococcus aureus Bacteremia infections (MRSAB). METHODS: A systematic overview was conducted by searching PubMed, Oxford academic, and Cochrane library up to June 2020. STUDY SELECTION AND DATA EXTRACTION: All English- language clinical trials, in vitro studies, and case reports related to the synergistic drug therapy for MRSAB. RESULTS: In the case of MRSAB infections, we examined two different in vitro studies that showed effective synergism with DAP and CFT. The Minimum Inhibitory Concentration (MIC) range observed for each is as follow: DAP 0.125-1 mg/L, CFT 0.38-1 mg/L, DAP + CFT 0.094-0.5 mg/L, vancomycin (VAN) 0.75-2 mg/L, VAN + CFT 0.25-2 mg/L. DAP + CFT combination displayed the most efficacy with the lowest MIC. The statistical analysis performed showed that DAP + CFT obtained significantly lower fractional inhibitory concentration (FIC) values (0.941 ± 0.328) compared with VAN + CFT. In vitro activities of regimens tested on DAP non-susceptibility and VAN intermediate after 96 hours showed DAP 8.29 ± 0.03a log10 CFU/mL, VAN 6.82 ± 0.04a log10 Colony Forming Unit (CFU)/mL, CFT 4.63 ± 0.19a log10 CFU/mL, DAP + CFT 1.15 ± 0.20b log10 CFU/mL, VAN + CFT 3.18 ± 0.49a log10 CFU/mL. (a meaning significantly different than DAP plus CFT, P< equal to 0.001b meaning therapeutic enhancement combination was defined as ≥ 2 log10 CFU/ml reduction over the most active single agent). Based on these results, although DAP was not susceptible, the Colony Forming Unit (CFU) for DAP and CFT had the best therapeutic results. CONCLUSION: In vitro studies have shown that a combination DAP and CFT is more efficacious than the combination of VAN and CFT in MRSA bacteremia infections. The synergic effects of DAP (bactericidal) and CFT (bactericidal) is statistically significant, in recent trials, warranting promising evidence for its use in complicated bacteremia infection.

Evaluation of Microparticulate (S)-4,5-Dihydroxy-2,3-pentanedione (DPD) as a Potential Vaccine Adjuvant.

Adjuvants potentiate the immune response against co-inoculated antigens in the vaccine formulation. Based on the mechanism of action, the adjuvants are classified as immunostimulatory adjuvants and vaccine delivery systems. (S)-4,5-Dihydroxy-2,3-pentanedione (DPD) is the precursor of bacterial quorum sensing molecule, autoinducer (AI)-2. We tested the immunogenicity and adjuvant potential of microparticulate formulation of (S)-DPD via in vitro evaluation. By formulating the microparticles of (S)-DPD, we consolidated the advantages of both the classes of adjuvants. The microparticulate (S)-DPD was tested for its immunogenicity and cytotoxicity. We further tested its adjuvant effect by combining it with particulate vaccines for measles and gonorrhea and compared the adjuvant effect observed with the microparticulate formulations of the FDA-approved adjuvants alum, MPL A®, and MF59®. Microparticulate (S)-DPD was found to be non-cytotoxic towards the antigen-presenting cells and had an adjuvant effect with microparticulate gonorrhea vaccine. Further studies with additional bacterial vaccines and the in vivo evaluation will confirm the potential of microparticulate (S)-DPD as a probable vaccine adjuvant candidate.

Potential Applications of Microparticulate-Based Bacterial Outer Membrane Vesicles (OMVs) Vaccine Platform for Sexually Transmitted Diseases (STDs): Gonorrhea, Chlamydia, and Syphilis.

Sexually transmitted diseases (STDs) are a major global health issue. Approximately 250 million new cases of STDs occur each year globally. Currently, only three STDs (human papillomavirus (HPV), hepatitis A, and hepatitis B) are preventable by vaccines. Vaccines for other STDs, including gonorrhea, chlamydia, and syphilis, await successful development. Currently, all of these STDs are treated with antibiotics. However, the efficacy of antibiotics is facing growing challenge due to the emergence of bacterial resistance. Therefore, alternative therapeutic approaches, including the development of vaccines against these STDs, should be explored to tackle this important global public health issue. Mass vaccination could be more efficient in reducing the spread of these highly contagious diseases. Bacterial outer membrane vesicle (OMV) is a potential antigen used to prevent STDs. OMVs are released spontaneously during growth by many Gram-negative bacteria. They present a wide range of surface antigens in native conformation that possess interesting properties such as immunogenicity, adjuvant potential, and the ability to be taken up by immune cells, all of which make them an attractive target for application as vaccines against pathogenic bacteria. The major challenge associated with the use of OMVs is its fragile structure and stability. However, a particulate form of the vaccine could be a suitable delivery system that can protect the antigen from degradation by a harsh acidic or enzymatic environment. The particulate form of the vaccine can also act as an adjuvant by itself. This review will highlight some practical methods for formulating microparticulate OMV-based vaccines for STDs.

Unexpected furanose-pyranose isomerization of 4-C-alkylribofuranose.

In this work, the attempt to synthesize 4-C-alkyl-S-ribosylhomocysteine (4-C-alkyl-SRH) analogues led to an unexpected and interesting intramolecular side reaction that occurred during the synthesis process. The detection of the compound 4-C-hexylribopyranose was confirmed by the use of 1H NMR and COSY spectra. The same reaction conditions were conducted for 4-C-octylribofuranose and led to the respective and 4-C-octylribopyranose. The 4-C-hexylribofuranose and 4-C-octylribofuranose were shown to exist in equilibrium with a ratio of 1:1 with their respective analogues 4-C-hexylribopyranose and 4-C-octylribopyranose.

A Mini-Review on Ceftaroline in Bacteremia Patients with Methicillin-Resistant Staphylococcus aureus (MRSA) Infections.

OBJECTIVE: The objective of this review is to describe the outcomes of patients treated with ceftaroline in the non-Food and Drug Administration (FDA) approved indication of methicillin-resistant Staphylococcus aureus (MRSA) infections in both pediatric and adult populations. DATA SOURCES: A systematic overview was conducted by searching PubMed, Medline, and The Cochrane Library up to January 2019. STUDY SELECTION AND DATA EXTRACTION: All English-language clinical trials and case reports related to the efficacy of ceftaroline in new, not-yet-approved FDA indications in MRSA infections in pediatric or adult populations. DATA SYNTHESIS: In the case of MRSA bacteremia (MRSAB) infections, three different randomized studies in pediatric patients showed effectiveness of ceftaroline. When used in the case of adult populations with MRSA bacteremia, a small trial of 16 patients showed 50% clinical success in patients with acute bacterial skin and skin structure infections versus 63% clinical success in patients with community-acquired bacterial pneumonia. Another case series of six refractory case reports showed 50% clinical success of ceftaroline in patients with MRSA. CONCLUSIONS: Although there are few case reports and limited data to date, ceftaroline fosamil should continue to be studied as an alternative therapy in MRSA infections in both pediatric and adult populations. Clinical success rates of ceftaroline were, in most cases, considered high when treating patients with MRSA infection. More clinical trials need to be studied. In the specific case of MRSA bacteremia, the treatment options remain few and ceftaroline should be extensively studied for the salvage treatment of MRSAB.

Buccal cell DNA extraction: yield, purity, and cost: a comparison of two methods.

Simple and cost-effective methods are needed to extract DNA in order to use it in large-scale studies. Blood is an excellent DNA source; however, it is costly and invasive thus an alternative is needed. Several kits and chemical protocols using buccal cells have been proposed for DNA extraction. The objective of the study is to evaluate buccal NaOH chemical protocol and Nucleospin Tissue Kit (BD Biosciences, Macery-Nagel, Germany) for DNA extraction. DNA swab samples were collected from 300 voluntary participants. DNA yields and purity were measured by NaOH and Nucleospin Tissue Kit techniques; the cost and time consumption for DNA extraction per sample were assessed as well. Results have shown that DNA amount and purity extracted by NaOH procedure was compared to that of the kit (p = 0.164; p = 0.249, respectively). NaOH method was considered cheaper and less time consuming (0.06 versus 3.80 USD, and 1.33 versus 3.59 minutes per sample, p < 0.001). Buccal cell derived DNA extracted by NaOH protocol can be considered a feasible substitute for more expensive and time-consuming kits.

S-Ribosylhomocysteine analogs containing a [4-thio]ribose ring.

The [4-thio]-S-ribosylhomocysteine (SRH) analogs containing substitution of a sulfur atom for the endocyclic oxygen were synthesized by coupling of the 4-thioribose substrates with a thiolate generated from the protected homocysteine. Coupling of the protected 1-deoxy-5-O-mesyl-S-oxo-4-thio-D-ribofuranose with homocysteinate salt gave the C4 epimers of [4-thio]-SRH at the sulfoxide oxidation level lacking a hydroxyl group at anomeric carbon. Treatment of these sulfoxides with BF3⋅Et2O/NaI affected simultaneous reduction to sulfide and global deprotection affording 1-deoxy-4-thio-SRH analog. Treatment of the protected 1-deoxy-S-oxo-4-thio-D-ribofuranose sulfoxide with DAST/SbCl3 resulted in the fluoro-Pummerer rearrangement to give 4-thio-ß-D-ribofuranosyl fluoride. Mesylation of the latter at 5-hydroxyl position followed by coupling with homocysteinate salt and subsequent global deprotection with trifluoroacetic acid afforded [4-thio]-SRH thiohemiacetal.