Replenishing the malaria drug discovery pipeline: Screening and hit evaluation of the MMV Hit Generation Library 1 (HGL1) against asexual blood stage Plasmodium falciparum, using a nano luciferase reporter read-out.

A central challenge of antimalarial therapy is the emergence of resistance to the components of artemisinin-based combination therapies (ACTs) and the urgent need for new drugs acting through novel mechanism of action. Over the last decade, compounds identified in phenotypic high throughput screens (HTS) have provided the starting point for six candidate drugs currently in the Medicines for Malaria Venture (MMV) clinical development portfolio. However, the published screening data which provided much of the new chemical matter for malaria drug discovery projects have been extensively mined. Here we present a new screening and selection cascade for generation of hit compounds active against the blood stage of Plasmodium falciparum. In addition, we validate our approach by testing a library of 141,786 compounds not reported earlier as being tested against malaria. The Hit Generation Library 1 (HGL1) was designed to maximise the chemical diversity and novelty of compounds with physicochemical properties associated with potential for further development. A robust HTS cascade containing orthogonal efficacy and cytotoxicity assays, including a newly developed and validated nanoluciferase-based assay was used to profile the compounds. 75 compounds (Screening Active hit rate of 0.05%) were identified meeting our stringent selection criteria of potency in drug sensitive (NF54) and drug resistant (Dd2) parasite strains (IC50 ≤ 2 µM), rapid speed of action and cell viability in HepG2 cells (IC50 ≥ 10 µM). Following further profiling, 33 compounds were identified that meet the MMV Confirmed Active profile and are high quality starting points for new antimalarial drug discovery projects.

Discovery of a First-In-Class Small Molecule Antagonist against the Adrenomedullin-2 Receptor: Structure-Activity Relationships and Optimization.

Class B G-protein-coupled receptors (GPCRs) remain an underexploited target for drug development. The calcitonin receptor (CTR) family is particularly challenging, as its receptors are heteromers comprising two distinct components: the calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) together with one of three accessory proteins known as receptor activity-modifying proteins (RAMPs). CLR/RAMP1 forms a CGRP receptor, CLR/RAMP2 forms an adrenomedullin-1 (AM1) receptor, and CLR/RAMP3 forms an adrenomedullin-2 (AM2) receptor. The CTR/RAMP complexes form three distinct amylin receptors. While the selective blockade of AM2 receptors would be therapeutically valuable, inhibition of AM1 receptors would cause clinically unacceptable increased blood pressure. We report here a systematic study of structure-activity relationships that has led to the development of first-in-class AM2 receptor antagonists. These compounds exhibit therapeutically valuable properties with 1000-fold selectivity over the AM1 receptor. These results highlight the therapeutic potential of AM2 antagonists.

Discovery of a First-in-Class Potent Small Molecule Antagonist against the Adrenomedullin-2 Receptor.

The hormone adrenomedullin has both physiological and pathological roles in biology. As a potent vasodilator, adrenomedullin is critically important in the regulation of blood pressure, but it also has several roles in disease, of which its actions in cancer are becoming recognized to have clinical importance. Reduced circulating adrenomedullin causes increased blood pressure but also reduces tumor progression, so drugs blocking all effects of adrenomedullin would be unacceptable clinically. However, there are two distinct receptors for adrenomedullin, each comprising the same G protein-coupled receptor (GPCR), the calcitonin receptor-like receptor (CLR), together with a different accessory protein known as a receptor activity-modifying protein (RAMP). The CLR with RAMP2 forms an adrenomedullin-1 receptor, and the CLR with RAMP3 forms an adrenomedullin-2 receptor. Recent research suggests that a selective blockade of adrenomedullin-2 receptors would be therapeutically valuable. Here we describe the design, synthesis, and characterization of potent small-molecule adrenomedullin-2 receptor antagonists with 1000-fold selectivity over the adrenomedullin-1 receptor, although retaining activity against the CGRP receptor. These molecules have clear effects on markers of pancreatic cancer progression in vitro, drug-like pharmacokinetic properties, and inhibit xenograft tumor growth and extend life in a mouse model of pancreatic cancer. Taken together, our data support the promise of a new class of anticancer therapeutics as well as improved understanding of the pharmacology of the adrenomedullin receptors and other GPCR/RAMP heteromers.

Novel Amino-pyrazole Ureas with Potent In Vitro and In Vivo Antileishmanial Activity.

Visceral leishmaniasis is a severe parasitic disease that is one of the most neglected tropical diseases. Treatment options are limited, and there is an urgent need for new therapeutic agents. Following an HTS campaign and hit optimization, a novel series of amino-pyrazole ureas has been identified with potent in vitro antileishmanial activity. Furthermore, compound 26 shows high levels of in vivo efficacy (>90%) against Leishmania infantum, thus demonstrating proof of concept for this series.

Enhanced HTS hit selection via a local hit rate analysis.

The postprocessing of high-throughput screening (HTS) results is complicated by the occurrence of false positives (inactive compounds misidentified as active by the primary screen) and false negatives (active compounds misidentified as inactive by the primary screen). An activity cutoff is frequently used to select "active" compounds from HTS data; however, this approach is insensitive to both false positives and false negatives. An alternative method that can minimize the occurrence of these artifacts will increase the efficiency of hit selection and therefore lead discovery. In this work, rather than merely using the activity of a given compound, we look at the presence and absence of activity among all compounds in its "chemical space neighborhood" to give a degree of confidence in its activity. We demonstrate that this local hit rate (LHR) analysis method outperforms hit selection based on ranking by primary screen activity values across ten diverse high throughput screens, spanning both cell-based and biochemical assay formats of varying biology and robustness. On average, the local hit rate analysis method was approximately 2.3-fold and approximately 1.3-fold more effective in identifying active compounds and active chemical series, respectively, than selection based on primary activity alone. Moreover, when applied to finding false negatives, this method was 2.3-fold better than ranking by primary activity alone. In most cases, novel hit series were identified that would have otherwise been missed. Additional uses of and observations regarding this HTS analysis approach are also discussed.