Screening the medicines for Malaria Venture "Malaria Box" against the Plasmodium falciparum aminopeptidases, M1, M17 and M18

Malaria is a parasitic disease that remains a global health burden. The ability of the parasite to rapidly develop resistance to therapeutics drives an urgent need for the delivery of new drugs. The Medicines for Malaria Venture have compounds known for their antimalarial ac- tivity, but not necessarily the molecular targets. In this study, we assess the ability of the “MMV 400” compounds to inhibit the activity of three metalloaminopeptidases from Plasmo- dium falciparum, PfA-M1, PfA-M17 and PfM18 AAP. We have developed a multiplex assay system to allow rapid primary screening of compounds against all three metalloaminopepti- dases, followed by detailed analysis of promising compounds. Our results show that there were no PfM18AAP inhibitors, whereas two moderate inhibitors of the neutral aminopepti- dases PfA-M1 and PfA-M17 were identified. Further investigation through structure-activity relationship studies and molecular docking suggest that these compounds are competitive inhibitors with novel binding mechanisms, acting through either non-classical zinc coordina- tion or independently of zinc binding altogether. Although it is unlikely that inhibition of PfA- M1 and/or PfA-M17 is the primary mechanism responsible for the antiplasmodial activity re- ported for these compounds, their detailed characterization, as presented in this work, pave the way for their further optimization as a novel class of dual PfA-M1/PfA-M17 inhibitors uti- lising non-classical zinc binding groups

Potent dual inhibitors of Plasmodium falciparum M1 and M17 aminopeptidases through optimization of S1 pocket interactions

Malaria remains a global health problem, and though international efforts for treatment and eradication have made some headway, the emergence of drug-resistant parasites threatens this progress. Antimalarial therapeutics acting via novel mechanisms are urgently required. P. falciparum M1 and M17 are neutral aminopeptidases which are essential for parasite growth and development. Previous work in our group has identified inhibitors capable of dual inhibition of PfA-M1 and PfA-M17, and revealed further regions within the protease S1 pockets that could be exploited in the development of ligands with improved inhibitory activity. Herein, we report the structure-based design and synthesis of novel hydroxamic acid analogues that are capable of potent inhibition of both PfA-M1 and PfA-M17. Furthermore, the developed compounds potently inhibit Pf growth in culture, including the multi-drug resistant strain Dd2. The ongoing development of dual PfA-M1/PfA-M17 inhibitors continues to be an attractive strategy for the design of novel antimalarial therapeutics

Identifying and Prioritising Behaviours to Slow Antimicrobial Resistance

As a nation with relatively low levels of AMR, due to both community and agricultural stewardship, as well as geographical isolation, Australia is somewhat unique. As this advantage is being eroded, this project aimed to investigate the spectrum of human behaviours that could be modified in order to slow the spread of AMR, building upon the argument that doable actions are the best-targeted and least complex to change. We conducted a workshop with a panel of diverse interdisciplinary AMR experts (from sociology, microbiology, agriculture, veterinary medicine, health and government) and identified twelve behaviours that, if undertaken by the public, would slow the spread of AMR. These were then assessed by a representative sample of the public (285 Australians) for current participation, likelihood of future participation (likelihood) and perceived benefits that could occur if undertaken (perceived impact). An impact-likelihood matrix was used to identify four priority behaviours: do not pressure your doctor for antibiotics; contact council to find out where you can safely dispose of cleaning products with antimicrobial marketing; lobby supermarkets to only sell antibiotic free meat products; and return unused antibiotics to a pharmacy. Among a multitude of behavioural options, this study also highlights the importance of tailoring doable actions to local conditions, increasing community education, and emphasizing the lack of a one-size fits all approach to tackling this global threat

X-ray crystal structures of an orally available aminopeptidase inhibitor, Tosedostat, bound to anti-malarial drug targets PfA-M1 and PfA-M17

New anti-malarial treatments are desperately required to face the spread of drug resistant parasites. Inhibition of metalloaminopeptidases, PfA-M1 and PfA-M17, is a validated therapeutic strategy for treatment of Plasmodium falciparum malaria. Here, we describe the crystal structures of PfA-M1 and PfA-M17 bound to chemotherapeutic agent Tosedostat. The inhibitor occupies the enzymes' putative product egress channels in addition to the substrate binding pockets; however, adopts different binding poses when bound to PfA-M1 and PfA-M17. These findings will be valuable for the continued development of selective inhibitors of PfA-M1 and PfA-M17

Reductive evolution in outer membrane protein biogenesis has not compromised cell surface complexity in Helicobacter pylori

Helicobacter pylori is a gram-negative bacterial pathogen that chronically inhabits the human stomach. To survive and maintain advantage, it has evolved unique host-pathogen interactions mediated by Helicobacter-specific proteins in the bacterial outer membrane. These outer membrane proteins (OMPs) are anchored to the cell surface via a C-terminal β-barrel domain, which requires their assembly by the β-barrel assembly machinery (BAM). Here we have assessed the complexity of the OMP C-terminal β-barrel domains employed by H. pylori, and characterized the H. pyloriBAM complex. Around 50 Helicobacter-specific OMPs were assessed with predictive structural algorithms. The data suggest that H. pylori utilizes a unique β-barrel architecture that might constitute H. pylori-specific Type V secretions system. The structural and functional diversity in these proteins is encompassed by their extramembrane domains. Bioinformatic and biochemical characterization suggests that the low β-barrel-complexity requires only minimalist assembly machinery. The H. pylori proteins BamA and BamD associate to form a BAM complex, with features of BamA enabling an oligomerization that might represent a mechanism by which a minimalist BAM complex forms a larger, sophisticated machinery capable of servicing the outer membrane proteome of H. pylori

Dixon plots of 1/V (<i>y axis</i>) versus inhibitor concentration (<i>x axis</i>) for <i>Pf</i>A-M1 (top panel) and <i>Pf</i>A-M17 (bottom panel).

<p>For <i>Pf</i>A-M1, substrate concentrations were 20 μM (blue) and 40 μM (red); inhibitor concentration in μM. For <i>Pf</i>A-M17, substrate concentrations were 5 μM (magenta) and 10 μM (green); inhibitor concentration in μM or nM as indicated. The point of intersection (-<i>K</i>_i) is indicated as a dotted line. (A) <i>Pf</i>A-M1 and compound <b>4. (B)</b><i>Pf</i>A-M1 and compound <b>12. (C)</b><i>Pf</i>A-M17 and compound <b>4. (D)</b><i>Pf</i>A-M17 and compound <b>12</b></p

Data collection and refinement statistics.

<p>* After an additional round of refinement including TLS parameters.</p><p>Data collection and refinement statistics.</p

Total aminopeptidase activity in the presence of 12 virtual screen compounds.

<p>The graph shown indicates the percentage of total aminopeptidase activity in the presence of 1 mM of compound (as indicated). Bestatin was used as control.</p

X ray crystal structures of compound 12 bound to <i>Pf</i>A-M1 and <i>Pf</i>A-M17.

<p>(<b>A</b>) View from the top of the catalytic pocket, showing compound <b>12</b> and the main interactions in the <i>Pf</i>A-M1 active site (in green). (<b>B</b>) Structural alignment of compound <b>12</b> as experimentally determined (green), FlexX-based pose (in blue) and MVD-based pose (in pink). (<b>C</b>) View from the top of the catalytic pocket, showing compound <b>12</b> (green) and the main interactions in the <i>Pf</i>A-M17 active site. (<b>D</b>) Structural alignment of compound <b>12</b> as experimentally determined (green), and the most similar predicted pose (pink).</p