The Manufacturers' Perspective on World Health Organization Prequalification of In Vitro Diagnostics.

In vitro diagnostic devices (IVDs) help clinicians determine specific conditions, monitor therapeutic efficacy, and prevent drug resistance development. While stringent regulatory authorities (SRAs) regulate IVDs in most high-income countries, regulatory authorities in many low- and middle-income countries (LMICs) are nonexistent or do not enforce rigorous standards. In 2010, the World Health Organization established its Prequalification of In Vitro Diagnostics (PQDx) program to ensure "access to safe, appropriate and affordable" IVDs, especially in LMICs with little or no domestic regulatory frameworks, thereby reaching underserved populations. However, challenges in PQDx policies and procedures include an overloaded pipeline, timelines not publicly available, confusion about which products PQDx focuses on, perceived burden for documenting changes to prequalified products, overlap with SRA approvals, and uncertainty around long-term financing. PQDx can maximize its impact by considering the perspective of IVD manufacturers; similarly, IVD manufacturers should exercise adequate quality control over their submissions and associated processes.

relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data.

UNLABELLED: Nuclear magnetic resonance (NMR) is a powerful tool for observing the motion of biomolecules at the atomic level. One technique, the analysis of relaxation dispersion phenomenon, is highly suited for studying the kinetics and thermodynamics of biological processes. Built on top of the relax computational environment for NMR dynamics is a new dispersion analysis designed to be comprehensive, accurate and easy-to-use. The software supports more models, both numeric and analytic, than current solutions. An automated protocol, available for scripting and driving the graphical user interface (GUI), is designed to simplify the analysis of dispersion data for NMR spectroscopists. Decreases in optimization time are granted by parallelization for running on computer clusters and by skipping an initial grid search by using parameters from one solution as the starting point for another -using analytic model results for the numeric models, taking advantage of model nesting, and using averaged non-clustered results for the clustered analysis. AVAILABILITY AND IMPLEMENTATION: The software relax is written in Python with C modules and is released under the GPLv3+ license. Source code and precompiled binaries for all major operating systems are available from http://www.nmr-relax.com. CONTACT: edward@nmr-relax.com.

Combination of the CCL5-derived peptide R4.0 with different HIV-1 blockers reveals wide target compatibility and synergic cobinding to CCR5.

R4.0, a synthetic CCL5/RANTES-derived peptide, exerts potent anti-HIV-1 activity via its nonactivating interaction with CCR5, the major HIV-1 coreceptor. CCR5 chronic activation may promote undesirable inflammatory effects and enhance viral infection; thus, receptor antagonism is a necessary requisite. HIV-1 gp120, CCL5, and maraviroc dock on CCR5 by sharing two receptor sites: the N terminus and the second extracellular loop. In combination studies, R4.0, CCL5, and maraviroc exhibited concomitant interactions with CCR5 and promoted synergic inhibition of HIV-1 in acute-infection assays. Furthermore, various degrees of additive/synergic HIV-1 inhibition were observed when R4.0 was tested in combination with drugs and lead compounds directed toward different viral targets (gp120, gp41, reverse transcriptase, and protease). In combination with tenofovir, R4.0 provides cross-clade synergic inhibition of primary HIV-1 isolates. Remarkably, an in vitro-generated maraviroc-resistant R5 HIV-1 strain was inhibited by R4.0 comparably to the wild-type strain, suggesting the presence of viral resistance barriers similar to those reported for CCL5. Overall, R4.0 appears to be a promising lead peptide with potential for combination in anti-HIV-1 therapy and in microbicide development to prevent sexual HIV-1 transmission.

Novel approaches to enable equitable access to monoclonal antibodies in low- and middle-income countries.

Monoclonal antibodies (mAbs) are revolutionizing management of non-communicable diseases in high-income countries and are increasingly being advanced for a range of infectious diseases (IDs). However, access to existing mAbs is limited in low- and middle-income countries (LMICs), and investment in developing fit-for-purpose mAbs for IDs that disproportionately affect LMICs has been limited. Underlying these access barriers are systemic challenges, including a lack of commercial incentives to target LMIC markets and complexity in manufacturing and regulatory processes. Novel strategies are needed to overcome systemic access barriers for mAbs. We outline key areas where new approaches could address these barriers, based on a multistakeholder consultation in March 2023. Three disease-market archetypes are identified to guide thinking about business models tailored to different contexts. New business models are needed to incentivize development and manufacturing of ID mAbs and to ensure mAbs are optimized with a target product profile and cost of goods that enable use in diverse LMIC settings. Lessons can be applied from voluntary licensing strategies and product development partnerships that have shown success in catalysing development and affordable supply for a range of infectious diseases. Technology transfer will be key to expand LMIC research and manufacturing capacity and to enable sustainable and diversified supply. Improved market intelligence, demand aggregation mechanisms, and portfolio-based manufacturing models could be used to de-risk commercial investment and establish a sustainable manufacturing ecosystem for affordable mAbs. Novel regulatory approaches and robust technology transfer may reduce data requirements and timelines for biosimilar approvals. Trailblazer products, with coordinated "end-to-end" support from funders, can demonstrate proof of concept for pathways to accessible mAbs across a broader range of LMICs. Research funders; local, regional, global health agencies; and, private sector partners should commit to implementing innovative partnerships and end-to-end strategies that enable equitable access to mAbs for infectious diseases in LMICs.

Conservation of flexible residue clusters among structural and functional enzyme homologues.

Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins.

Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor.

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled ((2)H/(15)N/(13)C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1ß were assessed by surface plasmon resonance yielding K(D) values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.

Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 1: addressing configurational instability through scaffold modification.

The optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly that possess a labile stereocenter at C3 is described. Quaternization of the C3 position of compound 1 in order to prevent racemization gave compound 2, which was inactive in our capsid disassembly assay. A likely explanation for this finding was revealed by in silico analysis predicting a dramatic increase in energy of the bioactive conformation upon quaternization of the C3 position. Replacement of the C3 of the diazepine ring with a nitrogen atom to give the 1,5-dihydro-benzo[f][1,3,5]triazepine-2,4-dione analog 4 was well tolerated. Introduction of a rigid spirocyclic system at the C3 position gave configurationally stable 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione analog 5, which was able to access the bioactive conformation without a severe energetic penalty and inhibit capsid assembly. Preliminary structure-activity relationships (SAR) and X-ray crystallographic data show that knowledge from the 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly can be transferred to these new scaffolds.

Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 2: structure-activity relationships (SAR) of the C3-phenyl moiety.

Detailed structure-activity relationships of the C3-phenyl moiety that allow for the optimization of antiviral potency of a series of 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione inhibitors of HIV capsid (CA) assembly are described. Combination of favorable substitutions gave additive SAR and allowed for the identification of the most potent compound in the series, analog 27. Productive SAR also transferred to the benzotriazepine and spirobenzodiazepine scaffolds, providing a solution to the labile stereocenter at the C3 position. The molecular basis of how compound 27 inhibits mature CA assembly is rationalized using high-resolution structural information. Our understanding of how compound 27 may inhibit immature Gag assembly is also discussed.

Nucleotide competing reverse transcriptase inhibitors: discovery of a series of non-basic benzofurano[3,2-d]pyrimidin-2-one derived inhibitors.

A HTS screen led to the identification of a benzofurano[3,2-d]pyrimidin-2-one core structure which upon further optimization resulted in 1 as a potent HIV-1 nucleotide competing reverse transcriptase inhibitor (NcRTI). Investigation of the SAR at N-1 allowed significant improvements in potency and when combined with the incorporation of heterocycles at C-8 resulted in potent analogues not requiring a basic amine to achieve antiviral activity. Additional modifications at N-1 resulted in 33 which demonstrated excellent antiviral potency and improved physicochemical properties.

Identification of potent and orally bioavailable nucleotide competing reverse transcriptase inhibitors: in vitro and in vivo optimization of a series of benzofurano[3,2-d]pyrimidin-2-one derived inhibitors.

Recently, a new class of HIV reverse transcriptase (HIV-RT) inhibitors has been reported. The novel mechanism of inhibition by this class involves competitive binding to the active site of the RT enzyme and has been termed Nucleotide-Competing Reverse Transcriptase Inhibitors (NcRTIs). In this publication we describe the optimization of a novel benzofurano[3,2-d]pyrimidin-2-one series of NcRTIs. The starting point for the current study was inhibitor 2, which had high biochemical and antiviral potency but only moderate permeability in a Caco-2 assay and high B-to-A efflux, resulting in moderate rat bioavailability and low Cmax. We present herein the results and strategies we employed to optimize both the potency as well as the permeability, metabolic stability and pharmacokinetic profile of this series. One of the key observations of the present study was the importance of shielding polar functionality, at least in the context of the current chemotype, to enhance permeability. These studies led to the identification of inhibitors 39 and 45, which display sub-nanomolar antiviral potency in a p24 ELISA assay with significantly reduced efflux ratios (ratios <1.5). These inhibitors also display excellent rat pharmacokinetic profiles with high bioavailabilities and low clearance.

Identification of benzofurano[3,2-d]pyrimidin-2-ones, a new series of HIV-1 nucleotide-competing reverse transcriptase inhibitors.

Screening of our sample collection led to the identification of a set of benzofurano[3,2-d]pyrimidine-2-one hits acting as nucleotide-competing HIV-1 reverse transcriptase inhibitiors (NcRTI). Significant improvement in antiviral potency was achieved when substituents were introduced at positions N1, C4, C7 and C8 on the benzofuranopyrimidone scaffold. The series was optimized from low micromolar enzymatic activity against HIV-1 RT and no antiviral activity to low nanomolar antiviral potency. Further profiling of inhibitor 30 showed promising overall in vitro properties and also demonstrated that its potency was maintained against viruses resistant to the other major classes of HIV-1 RT inhibitors.

Negotiating public-health intellectual property licensing agreements to increase access to health technologies: an insider's story.

Public health voluntary licensing of intellectual property has successfully been applied to increase access to medicines in certain disease areas, producing health benefits and economic savings, particularly in low-income and middle-income countries. There is however limited understanding of the intricacies of the approach, the modalities by which it works in practice, its levers and the trade-offs made. Such knowledge may be critical in deciding what role licensing should have in pandemic preparedness and equitable access to health technologies more broadly. This paper examines the case for licensing, the considerations for balancing public health needs, the challenges of negotiations, and the processes for validating proposed agreements. No access mechanism is perfect, but evidence suggests that public-health licensing has an important role to play, although it remains underused. Understanding some of the realities, strengths, limitations and complexities of applying the model may help calibrate expectations and develop incentives to expand its applications.

Voluntary licensing of long-acting HIV prevention and treatment regimens: using a proven collaboration- and competition-based mechanism to rapidly expand at-scale, sustainable, quality-assured and affordable supplies in LMICs.

INTRODUCTION: Emerging long-acting (LA) prevention and treatment medicines, technologies and regimens could be game-changing for the HIV response, helping reach the ambitious goal of halting the epidemic by 2030. To attain this goal, the rapid expansion of at-scale, sustainable, quality-assured, and affordable supplies of LA HIV prevention and treatment products through accelerated and stronger competition, involving both originator and generic companies, will be essential. To do this, global health stakeholders should take advantage of voluntary licensing of intellectual property (IP) rights, such as through the United Nations-backed, not-for-profit Medicines Patent Pool, as a proven mechanism to support broad access to existing HIV medicines across low- and middle-income countries (LMICs). DISCUSSION: While voluntary licensing may unlock the possibility for generic competition to take place ahead of patent expiry, there are additional elements-of amplified importance for more complex LA HIV medicines-that need to be taken into consideration. This paper discusses 10 enablers of voluntary licensing of IP rights as a model to rapidly expand at-scale, sustainable, quality-assured, and affordable supplies of LA HIV prevention and treatment regimens in LMICs: Identifying promising LA technology platforms and drug formulations at an early developmental stage and engaging with patent holders Consolidating a multidisciplinary network and strengthening early-stage coordination and collaboration to foster innovation Embedding public health considerations in product design and delivery Building innovative partnerships for product development and commercialization Raising awareness of and creating demand for emerging LA products Estimating the market size, ensuring sufficient competition and protecting sustainability Using technology transfer and hands-on technical support to reduce product development timelines and costs Exploring de-risking mechanisms and financial incentives to support generic manufacturers Optimizing strategies for generic product development and regulatory filings Aligning and coordinating efforts of stakeholders across the value chain. CONCLUSIONS: Rapid access to emerging LA prevention and treatment regimens and technologies can be facilitated by voluntary licensing-catalyzed and supplemented by enabling collaborative and non-duplicative efforts of various other stakeholders. This can effectively lead to improved-accelerated and cheaper-access to quality-assured medicines for populations in LMICs.

Expanding access to biotherapeutics in low-income and middle-income countries through public health non-exclusive voluntary intellectual property licensing: considerations, requirements, and opportunities.

Biotherapeutics, such as recombinant proteins and monoclonal antibodies, have become mainstays of modern medicine as shown by their increasing number in the WHO Model List of Essential Medicines. However, despite frequently offering clinical advantages over standards of care, they remain largely out of reach for populations in low-income and middle-income countries (LMICs), partly because of high costs. Accordingly, the WHO Model List of Essential Medicines Expert Committee has requested that the Medicines Patent Pool explore intellectual property licensing to address this challenge. We therefore investigated how licensing could successfully improve affordability of and timely access to biotherapeutics in LMICs, by leveraging expert consultations, literature analysis, and internal technical knowledge. The key elements identified as relevant to support access to affordable biosimilars in LMICs through licensing include: prioritising potential biotherapeutic targets according to their potential for public health impact; supporting biosimilar product and clinical development (including through technology transfer to expedite regulatory approval); and facilitating biosimilars' entry and use in LMICs (by meeting procurement, supply chain, and health system requirements).

Synergistic applications of MD and NMR for the study of biological systems.

Modern biological sciences are becoming more and more multidisciplinary. At the same time, theoretical and computational approaches gain in reliability and their field of application widens. In this short paper, we discuss recent advances in the areas of solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations that were made possible by the combination of both methods, that is, through their synergistic use. We present the main NMR observables and parameters that can be computed from simulations, and how they are used in a variety of complementary applications, including dynamics studies, model-free analysis, force field validation, and structural studies.

The economic and public health impact of intellectual property licensing of medicines for low-income and middle-income countries: a modelling study.

BACKGROUND: Non-exclusive voluntary licensing that is access-oriented has been suggested as an option to increase access to medicines to address the COVID-19 pandemic. To date, there has been little research on the effect of licensing, mainly focused on economic and supply chain considerations, and not on the benefits in terms of health outcomes. We aimed to study the economic and health effect of voluntary licensing for medicines for HIV and hepatitis C virus (HCV) in low-income and middle-income countries (LMICs). METHODS: A robust modelling framework was created to examine the difference between scenarios, with (factual) and without (counterfactual) a Medicines Patent Pool (MPP) licence for two medicines, dolutegravir and daclatasvir. Data were obtained from MPP licensees, as well as a large number of external sources. The primary outcomes were the cost savings and health impact between scenarios with and without MPP licences across all LMICs. Through its licences, MPP had access to the volumes and prices of licensed generic products sold in all covered countries on a quarterly basis. These data informed the volumes, prices, and uptake for the past factual scenarios and were the basis for modelling the future factual scenarios. These scenarios were then compared with a set of counterfactual scenarios in the absence of the studied licences. FINDINGS: Cumulatively, between 2017 and 2032, the model's central assumptions predicted an additional uptake of 15·494 (range 14·406-15·494) million patient-years of dolutegravir-based HIV treatments, 151 839 (34 575-312 973) deaths averted, and US$3·074 (1·837-5·617) billion saved through the MPP licence compared with the counterfactual scenario. For daclatasvir-based HCV treatments, the cumulative effect from 2015 to 2026 was predicted to be an additional uptake of 428 244 (127 584-636 270) patients treated with daclatasvir, 4070 (225-6323) deaths averted, and $107·593 (30·377-121·284) million saved with the licence compared with the counterfactual scenario. INTERPRETATION: The chain of effects linking upstream licensing to downstream outcomes can be modelled. Accordingly, credible quantitative estimates of economic and health effects arising from access-oriented voluntary licensing were obtained based on assumptions that early generic competition leads to price reductions that influence procurement decisions and enable the faster and broader uptake of recommended medicines, with beneficial economic and health effects. FUNDING: Unitaid.

Pediatric COVID-19 Therapeutics: Seizing the Right Research and Development Opportunities to Accelerate Access for Children.

Children, although at lower risk of poor outcomes from COVID-19 relative to adults, still stand to benefit from therapeutic interventions. Understanding of COVID-19 clinical presentation and prognosis in children is essential to optimize therapeutic trials design. This perspective illustrates how to collectively accelerate pediatric COVID-19 therapeutic research and development, based on the experience of the Global Accelerator for Paediatric Formulations.

Structure and dynamics of Mycobacterium tuberculosis truncated hemoglobin N: insights from NMR spectroscopy and molecular dynamics simulations.

The potent nitric oxide dioxygenase (NOD) activity (trHbN-Fe²âº-O2 + (•)NO → trHbN-Fe³âº-OH2 + NO3⁻) of Mycobacterium tuberculosis truncated hemoglobin N (trHbN) protects aerobic respiration from inhibition by (•)NO. The high activity of trHbN has been attributed in part to the presence of numerous short-lived hydrophobic cavities that allow partition and diffusion of the gaseous substrates (•)NO and O2 to the active site. We investigated the relation between these cavities and the dynamics of the protein using solution NMR spectroscopy and molecular dynamics (MD). Results from both approaches indicate that the protein is mainly rigid with very limited motions of the backbone N-H bond vectors on the picoseconds-nanoseconds time scale, indicating that substrate diffusion and partition within trHbN may be controlled by side-chains movements. Model-free analysis also revealed the presence of slow motions (microseconds-milliseconds), not observed in MD simulations, for many residues located in helices B and G including the distal heme pocket Tyr33(B10). All currently known crystal structures and molecular dynamics data of truncated hemoglobins with the so-called pre-A N-terminal extension suggest a stable α-helical conformation that extends in solution. Moreover, a recent study attributed a crucial role to the pre-A helix for NOD activity. However, solution NMR data clearly show that in near-physiological conditions these residues do not adopt an α-helical conformation and are significantly disordered and that the helical conformation seen in crystal structures is likely induced by crystal contacts. Although this lack of order for the pre-A does not disagree with an important functional role for these residues, our data show that one should not assume an helical conformation for these residues in any functional interpretation. Moreover, future molecular dynamics simulations should not use an initial α-helical conformation for these residues in order to avoid a bias based on an erroneous initial structure for the N-termini residues. This work constitutes the first study of a truncated hemoglobin dynamics performed by solution heteronuclear relaxation NMR spectroscopy.

Discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly.

The discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly is described. Synthesis of analogs of the 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione hit established structure-activity relationships. Replacement of the enamine functionality of the hit series with either an imidazole or a pyrazole ring led to compounds that inhibited both capsid assembly and reverse transcriptase. Optimization of the bicyclic benzodiazepine scaffold to include a 3-phenyl substituent led to lead compound 48, a pure capsid assembly inhibitor with improved antiviral activity.