Synthesis of analogs of the Gwt1 inhibitor manogepix (APX001A) and in vitro evaluation against Cryptococcus spp.

Fosmanogepix (APX001) is a first-in-class prodrug molecule that is currently in Phase 2 clinical trials for invasive fungal infections. The active moiety manogepix (APX001A) inhibits the novel fungal protein Gwt1. Gwt1 catalyzes an early step in the GPI anchor biosynthesis pathway. Here we describe the synthesis and evaluation of 292 new and 24 previously described analogs that were synthesized using a series of advanced intermediates to allow for rapid analoging. Several compounds demonstrated significantly (8- to 32-fold) improved antifungal activity against both Cryptococcus neoformans and C. gattii as compared to manogepix. Further in vitro characterization identified three analogs with a similar preliminary safety and in vitro profile to manogepix and superior activity against Cryptococcus spp.

Evaluation of Resistance Development to the Gwt1 Inhibitor Manogepix (APX001A) in Candida Species.

Manogepix (MGX) targets the conserved fungal Gwt1 enzyme required for acylation of inositol early in the glycosylphosphatidylinositol biosynthesis pathway. The prodrug fosmanogepix is currently in clinical development for the treatment of invasive fungal infections. We determined that the median frequencies of spontaneous mutations conferring reduced susceptibility to MGX in Candida albicans, C. glabrata, and C. parapsilosis ranged from 3 × 10-8 to <1.85 × 10-8 Serial passage on agar identified mutants of C. albicans and C. parapsilosis with reduced susceptibility to MGX; however, this methodology did not result in C. glabrata mutants with reduced susceptibility. Similarly, serial passage in broth resulted in ≤2-fold changes in population MIC values for C. tropicalis, C. auris, and C. glabrata A spontaneous V163A mutation in the Gwt1 protein of C. glabrata and a corresponding C. albicans heterozygous V162A mutant were obtained. A C. glabrata V163A Gwt1 mutant generated using CRISPR, along with V162A and V168A mutants expressed in C. albicans and Saccharomyces cerevisiae Gwt1, respectively, all demonstrated reduced susceptibility to MGX versus control strains, suggesting the importance of this valine residue to MGX binding across different species. Cross-resistance to the three major classes of antifungals was evaluated, but no changes in susceptibility to amphotericin B or caspofungin were observed in any mutant. No change was observed in fluconazole susceptibility, with the exception of a single non-Gwt1 mutant, where a 4-fold increase in the fluconazole MIC was observed. MGX demonstrated a relatively low potential for resistance development, consistent with other approved antifungal agents and those in clinical development.

Identification of Novel Genes and Biological Pathways That Overlap in Infectious and Nonallergic Diseases of the Upper and Lower Airways Using Network Analyses.

Previous genetic studies on susceptibility to otitis media and airway infections have focused on immune pathways acting within the local mucosal epithelium, and outside of allergic rhinitis and asthma, limited studies exist on the overlaps at the gene, pathway or network level between the upper and lower airways. In this report, we compared [1] pathways identified from network analysis using genes derived from published genome-wide family-based and association studies for otitis media, sinusitis, and lung phenotypes, to [2] pathways identified using differentially expressed genes from RNA-sequence data from lower airway, sinus, and middle ear tissues, in particular cholesteatoma tissue compared to middle ear mucosa. For otitis media, a large number of genes (n = 1,806) were identified as differentially expressed between cholesteatoma and middle ear mucosa, which in turn led to the identification of 68 pathways that are enriched in cholesteatoma. Two differentially expressed genes CR1 and SAA1 overlap in middle ear, sinus, and lower airway samples and are potentially novel genes for otitis media susceptibility. In addition, 56 genes were differentially expressed in both tissues from the middle ear and either sinus or lower airways. Pathways that are common in upper and lower airway diseases, whether from published DNA studies or from our RNA-sequencing analyses, include chromatin organization/remodeling, endocytosis, immune system process, protein folding, and viral process. Taken together, our findings from genetic susceptibility and differential tissue expression studies support the hypothesis that the unified airway theory wherein the upper and lower respiratory tracts act as an integrated unit also applies to infectious and nonallergic airway epithelial disease. Our results may be used as reference for identification of genes or pathways that are relevant to upper and lower airways, whether common across sites, or unique to each disease.

In Vitro and In Vivo Evaluation of APX001A/APX001 and Other Gwt1 Inhibitors against Cryptococcus.

Cryptococcal meningitis (CM), caused primarily by Cryptococcus neoformans, is uniformly fatal if not treated. Treatment options are limited, especially in resource-poor geographical regions, and mortality rates remain high despite current therapies. Here we evaluated the in vitro and in vivo activity of several compounds, including APX001A and its prodrug, APX001, currently in clinical development for the treatment of invasive fungal infections. These compounds target the conserved Gwt1 enzyme that is required for the localization of glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins in fungi. The Gwt1 inhibitors had low MIC values, ranging from 0.004 µg/ml to 0.5 µg/ml, against both C. neoformans and C. gattii APX001A and APX2020 demonstrated in vitro synergy with fluconazole (fractional inhibitory concentration index, 0.37 for both). In a CM model, APX001 and fluconazole each alone reduced the fungal burden in brain tissue (0.78 and 1.04 log10 CFU/g, respectively), whereas the combination resulted in a reduction of 3.52 log10 CFU/g brain tissue. Efficacy, as measured by a reduction in the brain and lung tissue fungal burden, was also observed for another Gwt1 inhibitor prodrug, APX2096, where dose-dependent reductions in the fungal burden ranged from 5.91 to 1.79 log10 CFU/g lung tissue and from 7.00 and 0.92 log10 CFU/g brain tissue, representing the nearly complete or complete sterilization of lung and brain tissue at the higher doses. These data support the further clinical evaluation of this new class of antifungal agents for the treatment of CM.

A calcineurin antifungal strategy with analogs of FK506.

A novel antifungal strategy targeting the inhibition of calcineurin is described. To develop a calcineurin based inhibitor of pathogenic fungi, analogs of FK506 were synthesized that were able to permeate mammalian but not fungal cells. Antagonists in combination with FK506 were not immunosuppressive and retained antifungal activity in A. fumigatus. To reduce the dosage burden of the antagonist, murine oral PK was improved an order of magnitude relative to previous FK506 antagonists.

Drug Sales, Gender, and Risk: Notions of Risk From the Perspective of Gang-Involved Young Adults.

We examine gender and meanings of risk in interviews (2007-2010) with gang-involved young men and women (n = 253) engaged in illicit drug sales in San Francisco, California. The in-depth interviews from this NIDA-funded study were coded using the software NVivo to identify patterns and themes. We examine their interpretations of the risks of drug-selling and their narratives about gender differences in these risks. We find distinct discourses regarding the role of femininities and masculinities and male and female bodies in shaping risk as well as the nexus between gender, family, and risk for female drug sellers.

"A cool little buzz": alcohol intoxication in the dance club scene.

In recent years, there has been increasing concern about youthful "binge" drinking and intoxication. Yet the meaning of intoxication remains under-theorized. This paper examines intoxication in a young adult nightlife scene, using data from a 2005-2008 National Institute on Drug Abuse-funded project on Asian American youth and nightlife. Analyzing in-depth qualitative interview data with 250 Asian American young adults in the San Francisco area, we examine their narratives about alcohol intoxication with respect to sociability, stress, and fun, and their navigation of the fine line between being "buzzed" and being "wasted." Finally, limitations of the study and directions for future research are noted.

Personal FM systems for children with autism spectrum disorders (ASD) and/or attention-deficit hyperactivity disorder (ADHD): an initial investigation.

UNLABELLED: The goal of this initial investigation was to examine the potential benefit of a frequency modulation (FM) system for 11 children diagnosed with autism spectrum disorders (ASD), attention-deficit hyperactivity disorder (ADHD), or both disorders through measures of speech recognition performance in noise, observed classroom behavior, and teacher-rated educational risk and listening behaviors. Use of the FM system resulted in significant average improvements in speech recognition in noise for the children with ASD and ADHD as well as large effect sizes. When compared to typically functioning peers, children with ASD and ADHD had significantly poorer average speech recognition performance in noise without the FM system but comparable average performance when the FM system was used. Similarly, classroom observations yielded a significant increase in on-task behaviors and large effect sizes when the FM system was in use during two separate trial periods. Although teacher ratings on questionnaires showed no significant improvement in the average level of educational risk of participants, they did indicate significant improvement in average listening behaviors during two trial periods with the FM system. Given the significantly better speech recognition in noise, increased on-task behaviors, and improved teacher ratings of listening behaviors with the FM system, these devices may be a viable option for children who have ASD and ADHD in the classroom. However, an individual evaluation including audiological testing and a functional evaluation in the child's primary learning environment will be necessary to determine the benefit of an FM system for a particular student. LEARNING OUTCOMES: 1. The reader will be able to describe the potential benefit of FM systems for children with ASD and/or ADHD. 2. The reader will be able to identify on-task versus off-task listening behaviors in children with ASD and/or ADHD. 3. The reader will be able to explain the components of a successful pre-fit education program that may be necessary prior to fitting an FM system in children with ASD.

Consumption, drugs and style: Constructing intra-ethnic boundaries in Asian American youth cultures.

Based on 250 qualitative interviews with Asian American young men and women in the dance/club scenes in the San Francisco area, we examine the interplay between consumption, style and taste cultures with issues of ethnic identity, gender and acculturation. We explore the ways that consumption and taste markers (e.g. fashion, cars, music and drugs) are used to establish or negotiate symbolic boundaries between groups in this youth culture. The picture they paint of the dance scene is one less about cohesiveness and unity and more about divisions and boundaries, not only between but also significantly within ethnic groupings. The choice of drugs and ways of exhibiting intoxication are among the types of consumption that the young people drew upon to mark symbolic boundaries and establish identities. The young men and women in this study discuss a number of key boundaries in the scene, e.g. between FOBs and twinkies, between pretty boys and thugs, as they attempt to establish the cultural legitimacy of their own styles of Asian American identities.

"HOW ASIAN AM I?" ASIAN AMERICAN YOUTH CULTURES, DRUG USE, AND ETHNIC IDENTITY CONSTRUCTION.

This article analyzes the construction of ethnic identity in the narratives of 100 young Asian Americans in a dance club/rave scene. We examine how illicit drug use and other consuming practices shape their understanding of Asian American identities, finding three distinct patterns. The first presents a disjuncture between Asian American ethnicity and drug use, seeing their own consumption as exceptional. The second argues their drug consumption is a natural outgrowth of their Asian American identity, allowing them to navigate the liminal space they occupy in American society. The final group presents Asian American drug use as normalized and constructs identity through taste and lifestyle boundary markers within social contexts of the dance scenes. These three narratives share a sense of ethnicity as dynamic, provisional, and constructed, allowing us to go beyond the static, essentialist models of ethnic identity that underlie much previous research on ethnicity, immigration, and substance use.

Food in the family. Bringing young people back in.

This article analyzes eating and beliefs about family meals in the qualitative interview narratives of 30 "at-risk" gang-involved young women in the San Francisco Bay Area. We begin our examination of consumption practices with a study of households and identify three major types-extended, single-parent and blended. Within these households, food purchasing and consumption activities are varied, and in many cases, our respondents rely upon extended family members and non-kin relations for support. In examining eating within the family, we identify two sets of practices and meanings: eating alone, and eating with others. Eating alone is symbolic of independence from one's family of origin, or is the result of familial conflict at the dinner table; however, it does not necessarily change our respondents' eating patterns. Eating with others in the family remains important, and many of the young women value family meals, although there are significant obstacles to eating regularly with the entire nuclear family. Many of these young women play an important role in the purchasing and preparation of food for family members as well. This paper highlights the importance of understanding family eating practices from the perspective of young people in the family, whose contribution to family ingestive practices has tended to be underestimated in much of available research literature.

Young Mother (in the) Hood: Gang Girls' Negotiation of New Identities.

This article examines the experiences of young women in street gangs who become mothers. Drawing on qualitative interviews with 65 young women in the San Francisco, CA, Bay Area, we examine their narratives about the transition to motherhood. In particular, we focus on the ways these young women negotiate femininities and attempt to reconcile their identities as young mothers and gang girls-both stigmatized identities. For many of the young women, motherhood entails a retreat from the street and a renewed emphasis on time spent in the home. While many receive (financial and childcare) support from their families, this also often means a diminution of the autonomy they experienced while more heavily involved in the gang. Issues of respect and respectability remain important for the young women, but the dimensions on which these are based change.

"It's one of the better drugs to use": perceptions of cocaine use among gay and bisexual Asian American men.

Research on drug use among gay and bisexual men has primarily focused on examining the link between drug use- most notably, methamphetamine-sexual practices, and risk of HIV transmission. Drawing on in-depth qualitative data from 40 interviews with gay and bisexual Asian American men, we examine perceptions and meanings associated with cocaine use in the San Francisco Bay Area gay community. We found that the participants, in contrast to their negative perceptions of methamphetamine use, believed that cocaine enhanced sociability and was acceptable for use in most social situations. Furthermore, participants perceived little connection between cocaine use and risky sexual practices, emphasizing the drug's safety relative to other illicit substances. Based on these findings, we suggest that an increase in the favorability of cocaine use might be an unintended consequence of methamphetamine prevention campaigns targeting the gay community, with their emphasis on the harmful effects of drug use, unsafe sex, and HIV risk.

Embarking on large-scale qualitative research: reaping the benefits of mixed methods in studying youth, clubs and drugs.

Qualitative research is often conceptualized as inherently small-scale research, primarily conducted by a lone researcher enmeshed in extensive and long-term fieldwork or involving in-depth interviews with a small sample of 20 to 30 participants. In the study of illicit drugs, traditionally this has often been in the form of ethnographies of drug-using subcultures. Such small-scale projects have produced important interpretive scholarship that focuses on the culture and meaning of drug use in situated, embodied contexts. Larger-scale projects are often assumed to be solely the domain of quantitative researchers, using formalistic survey methods and descriptive or explanatory models.In this paper, however, we will discuss qualitative research done on a comparatively larger scale-with in-depth qualitative interviews with hundreds of young drug users. Although this work incorporates some quantitative elements into the design, data collection, and analysis, the qualitative dimension and approach has nevertheless remained central. Larger-scale qualitative research shares some of the challenges and promises of smaller-scale qualitative work including understanding drug consumption from an emic perspective, locating hard-to-reach populations, developing rapport with respondents, generating thick descriptions and a rich analysis, and examining the wider socio-cultural context as a central feature. However, there are additional challenges specific to the scale of qualitative research, which include data management, data overload and problems of handling large-scale data sets, time constraints in coding and analyzing data, and personnel issues including training, organizing and mentoring large research teams. Yet large samples can prove to be essential for enabling researchers to conduct comparative research, whether that be cross-national research within a wider European perspective undertaken by different teams or cross-cultural research looking at internal divisions and differences within diverse communities and cultures.

THE PATH AND PROMISE OF FATHERHOOD FOR GANG MEMBERS.

While an increase in research on criminal desistance has occurred in recent years, little research has been applied to the gang field. Using qualitative interview data, this article examines fatherhood as a potential turning point in the lives of 91 gang members in the San Francisco Bay Area. Fatherhood initiated important subjective and affective transformations that led to changes in outlook, priorities and future orientation. However, these subjective changes were not sufficient unless accompanied by two additional features: first, changes in the amount of time spent on the streets and, second, an ability to support oneself or one's family with legal income. Though fatherhood is no panacea, becoming a father did act as an important turning point toward desistance and motivator for change for some.

Asian American identity and drug consumption: from acculturation to normalization.

This article analyzes the relationship between substance use and ethnic identity in the narratives of 206 young Asian Americans in a dance club/rave scene. We examined the meaning of drug use and found three types of narratives invoked to explain their drug use. The first noted difficulties arising from their Asian American identities, the experience of culture clash, and stresses associated with acculturation and Americanization. The second viewed their drug consumption as unusual among Asian Americans and saw their drug use as indicative of the degree to which they have grown apart from Asian culture and toward white/American culture. The final group saw their identities as Asian Americans neither as drug users nor as Asian American drug users as problematic. Drug use was a normal, accepted, and mundane part of their leisure time, not something they viewed as problematic or unusual.