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Molecular mechanisms and design principles for promiscuous inhibitors to avoid drug resistance: lessons learned from HIV-1 protease inhibition.

ABSTRACT: Molecular recognition is central to biology and ranges from highly selective to broadly promiscuous. The ability to modulate specificity at will is particularly important for drug development, and discovery of mechanisms contributing to binding specificity is crucial for our basic understanding of biology and for applications in health care. In this study, we used computational molecular design to create a large dataset of diverse small molecules with a range of binding specificities. We then performed structural, energetic, and statistical analysis on the dataset to study molecular mechanisms of achieving specificity goals. The work was done in the context of HIV-1 protease inhibition and the molecular designs targeted a panel of wild-type and drug-resistant mutant HIV-1 protease structures. The analysis focused on mechanisms for promiscuous binding to bind robustly even to resistance mutants. Broadly binding inhibitors tended to be smaller in size, more flexible in chemical structure, and more hydrophobic in nature compared to highly selective ones. Furthermore, structural and energetic analyses illustrated mechanisms by which flexible inhibitors achieved binding; we found ligand conformational adaptation near mutation sites and structural plasticity in targets through torsional flips of asymmetric functional groups to form alternative, compensatory packing interactions or hydrogen bonds. As no inhibitor bound to all variants, we designed small cocktails of inhibitors to do so and discovered that they often jointly covered the target set through mechanistic complementarity. Furthermore, using structural plasticity observed in experiments, and potentially in simulations, is suggested to be a viable means of designing adaptive inhibitors that are promiscuous binders.

SUBMITTER: Shen Y

PROVIDER: S-EPMC4829108 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Since the emergence of HIV and AIDS in the early 1980s, the development of safe and effective therapies has accompanied a massive increase in our understanding of the fundamental processes that drive HIV biology. As basic HIV research has informed the development of novel therapies, HIV inhibitors have been used as probes for investigating basic mechanisms of HIV-1 replication, transmission, and pathogenesis. This positive feedback cycle has led to the development of highly effective combination antiretroviral therapy (cART), which has helped stall the progression to AIDS, prolong lives, and reduce transmission of the virus. However, to combat the growing rates of virologic failure and toxicity associated with long-term therapy, it is important to diversify our repertoire of HIV-1 treatments by identifying compounds that block additional steps not targeted by current drugs. Most of the available therapeutics disrupt early events in the replication cycle, with the exception of the protease (PR) inhibitors, which act at the virus maturation step. HIV-1 maturation consists of a series of biochemical changes that facilitate the conversion of an immature, noninfectious particle to a mature infectious virion. These changes include proteolytic processing of the Gag polyprotein by the viral protease (PR), structural rearrangement of the capsid (CA) protein, and assembly of individual CA monomers into hexamers and pentamers that ultimately form the capsid. Here, we review the development and therapeutic potential of maturation inhibitors (MIs), an experimental class of anti-HIV-1 compounds with mechanisms of action distinct from those of the PR inhibitors. We emphasize the key insights into HIV-1 biology and structure that the study of MIs has provided. We will focus on three distinct groups of inhibitors that block HIV-1 maturation: (1) compounds that block the processing of the CA-spacer peptide 1 (SP1) cleavage intermediate, the original class of compounds to which the term MI was applied; (2) CA-binding inhibitors that disrupt capsid condensation; and (3) allosteric integrase inhibitors (ALLINIs) that block the packaging of the viral RNA genome into the condensing capsid during maturation. Although these three classes of compounds have distinct structures and mechanisms of action, they share the ability to block the formation of the condensed conical capsid, thereby blocking particle infectivity.

Project description:BackgroundHistone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active 'probe' molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells.ResultsA panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition.ConclusionsHigh-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors.

Project description:An important pathogenetic distinction in the classification of myeloproliferative neoplasms (MPNs) is the presence or absence of the BCR-ABL fusion gene, which encodes a unique oncogenic tyrosine kinase. The BCR-ABL fusion, caused by the formation of the Philadelphia chromosome (Ph) through translocation, constitutes the disease-initiating event in chronic myeloid leukemia. The development of successive BCR-ABL-targeted tyrosine-kinase inhibitors has led to greatly improved outcomes in patients with chronic myeloid leukemia, including high rates of complete hematologic, cytogenetic, and molecular responses. Such levels of treatment success have long been elusive for patients with Ph-negative MPNs, because of the difficulties in identifying specific driver proteins suitable as drug targets. However, in recent years an improved understanding of the complex pathobiology of classic Ph-negative MPNs, characterized by variable, overlapping multimutation profiles, has prompted the development of better and more broadly targeted (to pathway rather than protein) treatment options, particularly JAK inhibitors. In classic Ph-negative MPNs, overactivation of JAK-dependent signaling pathways is a central pathogenic mechanism, and mutually exclusive mutations in JAK2, MPL, and CALR linked to aberrant JAK activation are now recognized as key drivers of disease progression in myelofibrosis (MF). In clinical trials, the JAK1/JAK2 inhibitor ruxolitinib - the first therapy approved for MF worldwide - improved disease-related splenomegaly and symptoms independent of JAK2 (V617F) mutational status, and prolonged survival compared with placebo or standard therapy in patients with advanced MF. In separate trials, ruxolitinib also provided comprehensive hematologic control in patients with another Ph-negative MPN - polycythemia vera. However, complete cytogenetic or molecular responses with JAK inhibitors alone are normally not observed, underscoring the need for novel combination therapies of JAK inhibitors and complementary agents that better address the complexity of the pathobiology of classic Ph-negative MPNs. Here, we discuss the role of tyrosine-kinase inhibitors in the current MPN-treatment landscape.

Project description:Self-management is critical to achieving diabetes treatment goals. Mobile phones and Bluetooth® can supportself-management and lifestyle changes for chronic diseases such as diabetes. A mobile health (mHealth) research platform--the Few Touch Application (FTA)--is a tool designed to support the self-management of diabetes. The FTA consists of a mobile phone-based diabetes diary, which can be updated both manually from user input and automatically by wireless data transfer, and which provides personalized decision support for the achievement of personal health goals. Studies and applications (apps) based on FTAs have included: (1) automatic transfer of blood glucose (BG) data; (2) short message service (SMS)-based education for type 1diabetes (T1DM); (3) a diabetes diary for type 2 diabetes (T2DM); (4) integrating a patient diabetes diary with health care (HC) providers; (5) a diabetes diary for T1DM; (6) a food picture diary for T1DM; (7) physical activity monitoring for T2DM; (8) nutrition information for T2DM; (9) context sensitivity in mobile self-help tools; and (10) modeling of BG using mobile phones. We have analyzed the performance of these 10 FTA-based apps to identify lessons for designing the most effective mHealth apps. From each of the 10 apps of FTA, respectively, we conclude: (1) automatic BG data transfer is easy to use and provides reassurance; (2) SMS-based education facilitates parent-child communication in T1DM; (3) the T2DM mobile phone diary encourages reflection; (4) the mobile phone diary enhances discussion between patients and HC professionals; (5) the T1DM mobile phone diary is useful and motivational; (6) the T1DM mobile phone picture diary is useful in identifying treatment obstacles; (7) the step counter with automatic data transfer promotes motivation and increases physical activity in T2DM; (8) food information on a phone for T2DM should not be at a detailed level; (9) context sensitivity has good prospects and is possible to implement on today's phones; and (10) BG modeling on mobile phones is promising for motivated T1DM users. We expect that the following elements will be important in future FTA designs: (A) automatic data transfer when possible; (B) motivational and visual user interfaces; (C) apps with considerable health benefits in relation to the effort required; (D) dynamic usage, e.g., both personal and together with HC personnel, long-/short-term perspective; and (E) inclusion of context sensitivity in apps. We conclude that mHealth apps will empower patients to take a more active role in managing their own health.

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Molecular mechanisms and design principles for promiscuous inhibitors to avoid drug resistance: lessons learned from HIV-1 protease inhibition.

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