Productive employment generation: an effective way to revitalize informal settlements in Pakistan

Dissertação de mestrado em Administração PúblicaOne of the major concerns of government trying to devise sustainable urban policies is rapidly growing informal settlements in and at outskirts of cities. The problem is highly prevalent in developing countries and needs urgent attention. Informal settlements are the most disadvantaged sections of the society that suffer extreme poverty conditions and are excluded from social, spatial and economic developments. The study is undertaken for Pakistan with the intention to find the best possible way through which quality of life of residents of informal settlements can be improved. Informal settlements from three provinces Punjab, Sindh and Khyber Pakhtunkhwa in Pakistan are scrutinized in detail to reveal the current conditions of these residents in terms of health, education, crime, social capital and happiness. The study shows the deviation between the life of affluent classes and the poor classes and touches the important subject of income inequality and its implications. It simultaneously explores a link between unemployment, underemployment, informal sector and informal settlements. The roles of the federal, provincial and local governments as policy makers and implementers are highlighted by studying urban policies adopted post partition of the subcontinent and the effectiveness of each policy is analyzed to understand their impact on the lives of these residents as well as on other civilians. Lastly, the importance of productive employment is examined in search of an effective long-term sustainable solution.Uma das principais preocupações do governo em relação a conceção de políticas urbanas sustentáveis é o rápido crescimento dos urbanizaçõesinformais nas cidades e nas periferias. O problema é particularmente prevalente em países em desenvolvimento e precisa de atenção urgente. As urbanizações informais são os setores mais desfavorecidos da sociedade sofrendo de condições de extrema pobreza e são excluídos do desenvolvimento social, espacial e econômico. O estudo é realizado no Paquistão, com a intenção de encontrar a melhor maneira possível de melhorar a qualidade de vida dos residentes de urbanizaçõesinformais. As urbanizaçõesinformais de três províncias, Punjab, Sindh e Khyber Pakhtunkhwa no Paquistão, são examinados em detalhes para revelar as condições atuais dosresidentes em termos de saúde, educação, crime, capital social e felicidade. O estudo mostra o desvio entre a vida das classes abastadas e das classes pobres e aborda o importante tema da desigualdade de rendimentoe suas implicações. Simultaneamente, explora uma ligação entre desemprego, subemprego, setor informal e as urbanizaçõesinformais. Os papéis dos governos federal, provincial e local como formuladores e implementadores de políticas são destacados pelo estudo das políticas urbanas adotadas após a partição do subcontinente. A eficácia de cada política é analisada para compreender o seu impacto nas vidas desses residentes, bem como nas outras esferas civis. Por fim, examina-se a importância do emprego produtivo na busca de uma solução eficaz e sustentável de longo prazo

Macrodomain binding compound MRS 2578 inhibits alphavirus replication

Peer reviewe

Macrodomain binding compound MRS 2578 inhibits alphavirus replication

Peer reviewe

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Multiple crystal forms of human MacroD2

Abstract MacroD2 is one of the three human macrodomain proteins characterized by their protein-linked mono-ADP-ribosyl-hydrolyzing activity. MacroD2 is a single-domain protein that contains a deep ADP-ribose-binding groove. In this study, new crystallization conditions for MacroD2 were found and three crystal structures of human MacroD2 in the apo state were solved in space groups P4₁2₁2, P43212 and P4₃, and refined at 1.75, 1.90 and 1.70 Å resolution, respectively. Structural comparison of the apo crystal structures with the previously reported crystal structure of MacroD2 in complex with ADP-ribose revealed conformational changes in the side chains of Val101, Ile189 and Phe224 induced by the binding of ADP-ribose in the active site. These conformational variations may potentially facilitate design efforts of a MacroD2 inhibitor

Activity-based screening assay for mono-ADP-Ribosylhydrolases

Abstract ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting β-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD⁺, anomer of β-NAD⁺, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD⁺ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolase

A molecular toolbox for ADP-ribosyl binding proteins

Summary Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or viral infections, making them attractive drug targets. We present a robust and accessible assay applicable to both hydrolyzing or non-hydrolyzing binders of mono- and poly-ADP-ribosyl groups. This technology relies on a C-terminal tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups or analogs. By fusing the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we generate robust FRET partners and confirm the interaction with 22 known ADP-ribosyl binders. The applicability for high-throughput screening of inhibitors is demonstrated with the SARS-CoV-2 nsp3 macrodomain, for which we identify suramin as a moderate-affinity yet non-specific inhibitor. High-affinity ADP-ribosyl binders fused to nanoluciferase complement this technology, enabling simple blot-based detection of ADP-ribosylated proteins. All these tools can be produced in Escherichia coli and will help in ADP-ribosylation research and drug discovery

Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe

Abstract Here, we describe a protocol to set up a screening assay for ADP-ribosyl binding proteins including proteins that possess O-glycosidase or N-glycosidase activities. The FRET-based assay measures the interaction of any ADP-ribosyl binding protein fused to CFP with a cysteine-ADP-ribosylated GAP-tag fused to YFP. Recombinant PtxS1 and PARP2 are used to mono-ADP-ribosylate and poly-ADP-ribosylate the GAP-tag. The protocol does not require specialized compounds or substrates, making it accessible and easy to adapt in any laboratory or for other proteins of interest. For complete details on the use and execution of this profile, please refer to Sowa et al. (2021)

Discovery of compounds that inhibit SARS-CoV-2 Mac1-ADP-ribose binding by high-throughput screening

Abstract The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen ∼38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC₅₀ values less than 100 μM, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors

Antimicrobial activity of aurein 2.5 against yeasts

Fungal infections with multiple resistance to conventional antifungals are increasingly becoming a medical problem, and there is an urgent need for new antifungal compounds with novel mechanisms of action. Here, we show that aurein 2.5, a naturally occurring peptide antibiotic, displays activity against the fungal strains: Rhodotorula rubra and Schizosaccharomyces pombe (MICs 65%) in the presence of lipid membranes derived from these organisms and showed strong propensities to penetrate (π ≥ 13 mN m-1) and lyse them (> 70%). Based on these data, we suggest that aurein 2.5 kills yeasts via membranolytic mechanisms and may act as a template for the development of therapeutically useful antifungal agents