The Immune Response to Mycobacterium tuberculosis in HIV-1-Coinfected Persons.

Globally, about 36.7 million people were living with HIV infection at the end of 2015. The most frequent infection co-occurring with HIV-1 is Mycobacterium tuberculosis-374,000 deaths per annum are attributable to HIV-tuberculosis, 75% of those occurring in Africa. HIV-1 infection increases the risk of tuberculosis by a factor of up to 26 and alters its clinical presentation, complicates diagnosis and treatment, and worsens outcome. Although HIV-1-induced depletion of CD4+ T cells underlies all these effects, more widespread immune deficits also contribute to susceptibility and pathogenesis. These defects present a challenge to understand and ameliorate, but also an opportunity to learn and optimize mechanisms that normally protect people against tuberculosis. The most effective means to prevent and ameliorate tuberculosis in HIV-1-infected people is antiretroviral therapy, but this may be complicated by pathological immune deterioration that in turn requires more effective host-directed anti-inflammatory therapies to be derived.

The Art and Science of Molecular Docking.

Molecular docking has become an essential part of a structural biologist's and medicinal chemist's toolkits. Given a chemical compound and the three-dimensional structure of a molecular target-for example, a protein-docking methods fit the compound into the target, predicting the compound's bound structure and binding energy. Docking can be used to discover novel ligands for a target by screening large virtual compound libraries. Docking can also provide a useful starting point for structure-based ligand optimization or for investigating a ligand's mechanism of action. Advances in computational methods, including both physics-based and machine learning approaches, as well as in complementary experimental techniques, are making docking an even more powerful tool. We review how docking works and how it can drive drug discovery and biological research. We also describe its current limitations and ongoing efforts to overcome them.

Pan-cancer proteogenomics expands the landscape of therapeutic targets.

Fewer than 200 proteins are targeted by cancer drugs approved by the Food and Drug Administration (FDA). We integrate Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomics data from 1,043 patients across 10 cancer types with additional public datasets to identify potential therapeutic targets. Pan-cancer analysis of 2,863 druggable proteins reveals a wide abundance range and identifies biological factors that affect mRNA-protein correlation. Integration of proteomic data from tumors and genetic screen data from cell lines identifies protein overexpression- or hyperactivation-driven druggable dependencies, enabling accurate predictions of effective drug targets. Proteogenomic identification of synthetic lethality provides a strategy to target tumor suppressor gene loss. Combining proteogenomic analysis and MHC binding prediction prioritizes mutant KRAS peptides as promising public neoantigens. Computational identification of shared tumor-associated antigens followed by experimental confirmation nominates peptides as immunotherapy targets. These analyses, summarized at https://targets.linkedomics.org, form a comprehensive landscape of protein and peptide targets for companion diagnostics, drug repurposing, and therapy development.

Direct interrogation of context-dependent GPCR activity with a universal biosensor platform.

G protein-coupled receptors (GPCRs) are the largest family of druggable proteins encoded in the human genome, but progress in understanding and targeting them is hindered by the lack of tools to reliably measure their nuanced behavior in physiologically relevant contexts. Here, we developed a collection of compact ONE vector G-protein Optical (ONE-GO) biosensor constructs as a scalable platform that can be conveniently deployed to measure G-protein activation by virtually any GPCR with high fidelity even when expressed endogenously in primary cells. By characterizing dozens of GPCRs across many cell types like primary cardiovascular cells or neurons, we revealed insights into the molecular basis for G-protein coupling selectivity of GPCRs, pharmacogenomic profiles of anti-psychotics on naturally occurring GPCR variants, and G-protein subtype signaling bias by endogenous GPCRs depending on cell type or upon inducing disease-like states. In summary, this open-source platform makes the direct interrogation of context-dependent GPCR activity broadly accessible.

Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

First-in-class MKK4 inhibitors enhance liver regeneration and prevent liver failure.

Diminished hepatocyte regeneration is a key feature of acute and chronic liver diseases and after extended liver resections, resulting in the inability to maintain or restore a sufficient functional liver mass. Therapies to restore hepatocyte regeneration are lacking, making liver transplantation the only curative option for end-stage liver disease. Here, we report on the structure-based development and characterization (nuclear magnetic resonance [NMR] spectroscopy) of first-in-class small molecule inhibitors of the dual-specificity kinase MKK4 (MKK4i). MKK4i increased liver regeneration upon hepatectomy in murine and porcine models, allowed for survival of pigs in a lethal 85% hepatectomy model, and showed antisteatotic and antifibrotic effects in liver disease mouse models. A first-in-human phase I trial (European Union Drug Regulating Authorities Clinical Trials [EudraCT] 2021-000193-28) with the clinical candidate HRX215 was conducted and revealed excellent safety and pharmacokinetics. Clinical trials to probe HRX215 for prevention/treatment of liver failure after extensive oncological liver resections or after transplantation of small grafts are warranted.

Structural mechanisms of α7 nicotinic receptor allosteric modulation and activation.

The α7 nicotinic acetylcholine receptor is a pentameric ligand-gated ion channel that plays an important role in cholinergic signaling throughout the nervous system. Its unique physiological characteristics and implications in neurological disorders and inflammation make it a promising but challenging therapeutic target. Positive allosteric modulators overcome limitations of traditional α7 agonists, but their potentiation mechanisms remain unclear. Here, we present high-resolution structures of α7-modulator complexes, revealing partially overlapping binding sites but varying conformational states. Structure-guided functional and computational tests suggest that differences in modulator activity arise from the stable rotation of a channel gating residue out of the pore. We extend the study using a time-resolved cryoelectron microscopy (cryo-EM) approach to reveal asymmetric state transitions for this homomeric channel and also find that a modulator with allosteric agonist activity exploits a distinct channel-gating mechanism. These results define mechanisms of α7 allosteric modulation and activation with implications across the pentameric receptor superfamily.

Discovery of natural-product-derived sequanamycins as potent oral anti-tuberculosis agents.

The emergence of drug-resistant tuberculosis has created an urgent need for new anti-tubercular agents. Here, we report the discovery of a series of macrolides called sequanamycins with outstanding in vitro and in vivo activity against Mycobacterium tuberculosis (Mtb). Sequanamycins are bacterial ribosome inhibitors that interact with the ribosome in a similar manner to classic macrolides like erythromycin and clarithromycin, but with binding characteristics that allow them to overcome the inherent macrolide resistance of Mtb. Structures of the ribosome with bound inhibitors were used to optimize sequanamycin to produce the advanced lead compound SEQ-9. SEQ-9 was efficacious in mouse models of acute and chronic TB as a single agent, and it demonstrated bactericidal activity in a murine TB infection model in combination with other TB drugs. These results support further investigation of this series as TB clinical candidates, with the potential for use in new regimens against drug-susceptible and drug-resistant TB.

Rapid adaptation to CDK2 inhibition exposes intrinsic cell-cycle plasticity.

CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.

Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors.

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.

Multi-chamber cardioids unravel human heart development and cardiac defects.

The number one cause of human fetal death are defects in heart development. Because the human embryonic heart is inaccessible and the impacts of mutations, drugs, and environmental factors on the specialized functions of different heart compartments are not captured by in vitro models, determining the underlying causes is difficult. Here, we established a human cardioid platform that recapitulates the development of all major embryonic heart compartments, including right and left ventricles, atria, outflow tract, and atrioventricular canal. By leveraging 2D and 3D differentiation, we efficiently generated progenitor subsets with distinct first, anterior, and posterior second heart field identities. This advance enabled the reproducible generation of cardioids with compartment-specific in vivo-like gene expression profiles, morphologies, and functions. We used this platform to unravel the ontogeny of signal and contraction propagation between interacting heart chambers and dissect how mutations, teratogens, and drugs cause compartment-specific defects in the developing human heart.

QKI shuttles internal m7G-modified transcripts into stress granules and modulates mRNA metabolism.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 5' cap or within tRNAs/rRNAs, also exists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)" motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

Validating Small Molecule Chemical Probes for Biological Discovery.

Small molecule chemical probes are valuable tools for interrogating protein biological functions and relevance as a therapeutic target. Rigorous validation of chemical probe parameters such as cellular potency and selectivity is critical to unequivocally linking biological and phenotypic data resulting from treatment with a chemical probe to the function of a specific target protein. A variety of modern technologies are available to evaluate cellular potency and selectivity, target engagement, and functional response biomarkers of chemical probe compounds. Here, we review these technologies and the rationales behind using them for the characterization and validation of chemical probes. In addition, large-scale phenotypic characterization of chemical probes through chemical genetic screening is increasingly leading to a wealth of information on the cellular pharmacology and disease involvement of potential therapeutic targets. Extensive compound validation approaches and integration of phenotypic information will lay foundations for further use of chemical probes in biological discovery.

In situ identification of cellular drug targets in mammalian tissue.

The lack of tools to observe drug-target interactions at cellular resolution in intact tissue has been a major barrier to understanding in vivo drug actions. Here, we develop clearing-assisted tissue click chemistry (CATCH) to optically image covalent drug targets in intact mammalian tissues. CATCH permits specific and robust in situ fluorescence imaging of target-bound drug molecules at subcellular resolution and enables the identification of target cell types. Using well-established inhibitors of endocannabinoid hydrolases and monoamine oxidases, direct or competitive CATCH not only reveals distinct anatomical distributions and predominant cell targets of different drug compounds in the mouse brain but also uncovers unexpected differences in drug engagement across and within brain regions, reflecting rare cell types, as well as dose-dependent target shifts across tissue, cellular, and subcellular compartments that are not accessible by conventional methods. CATCH represents a valuable platform for visualizing in vivo interactions of small molecules in tissue.

Structural basis for the severe adverse interaction of sofosbuvir and amiodarone on L-type Cav channels.

Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Cav channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Cav1.1 and Cav1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Cav channels.

Molecular recognition of morphine and fentanyl by the human µ-opioid receptor.

Morphine and fentanyl are among the most used opioid drugs that confer analgesia and unwanted side effects through both G protein and arrestin signaling pathways of µ-opioid receptor (µOR). Here, we report structures of the human µOR-G protein complexes bound to morphine and fentanyl, which uncover key differences in how they bind the receptor. We also report structures of µOR bound to TRV130, PZM21, and SR17018, which reveal preferential interactions of these agonists with TM3 side of the ligand-binding pocket rather than TM6/7 side. In contrast, morphine and fentanyl form dual interactions with both TM3 and TM6/7 regions. Mutations at the TM6/7 interface abolish arrestin recruitment of µOR promoted by morphine and fentanyl. Ligands designed to reduce TM6/7 interactions display preferential G protein signaling. Our results provide crucial insights into fentanyl recognition and signaling of µOR, which may facilitate rational design of next-generation analgesics.

State-selective modulation of heterotrimeric Gαs signaling with macrocyclic peptides.

The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, with the exception of the Gα subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 1012 cyclic peptides to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gαs, respectively. Both macrocyclic peptides fine-tune Gαs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/α3 pocket. Cell-permeable analogs of GN13 and GD20 modulate Gαs/Gßγ signaling in cells through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gαs provides a path for further development of state-dependent GTPase inhibitors.

A general chemical principle for creating closure-stabilizing integrin inhibitors.

Integrins are validated drug targets with six approved therapeutics. However, small-molecule inhibitors to three integrins failed in late-stage clinical trials for chronic indications. Such unfavorable outcomes may in part be caused by partial agonism, i.e., the stabilization of the high-affinity, extended-open integrin conformation. Here, we show that the failed, small-molecule inhibitors of integrins αIIbß3 and α4ß1 stabilize the high-affinity conformation. Furthermore, we discovered a simple chemical feature present in multiple αIIbß3 antagonists that stabilizes integrins in their bent-closed conformation. Closing inhibitors contain a polar nitrogen atom that stabilizes, via hydrogen bonds, a water molecule that intervenes between a serine residue and the metal in the metal-ion-dependent adhesion site (MIDAS). Expulsion of this water is a requisite for transition to the open conformation. This change in metal coordination is general to integrins, suggesting broad applicability of the drug-design principle to the integrin family, as validated with a distantly related integrin, α4ß1.

Cannabinoid receptor 1 antagonist genistein attenuates marijuana-induced vascular inflammation.

Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, binds to cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis was an risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD. In silico virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells were used to model Δ9-THC-induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference, and genistein attenuated the effects of Δ9-THC. In mice, genistein blocked Δ9-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system. Genistein is a CB1 antagonist that attenuates Δ9-THC-induced atherosclerosis.

Structural basis of γ-secretase inhibition and modulation by small molecule drugs.

Development of γ-secretase inhibitors (GSIs) and modulators (GSMs) represents an attractive therapeutic opportunity for Alzheimer's disease (AD) and cancers. However, how these GSIs and GSMs target γ-secretase has remained largely unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human γ-secretase bound individually to two GSI clinical candidates, Semagacestat and Avagacestat, a transition state analog GSI L685,458, and a classic GSM E2012, at overall resolutions of 2.6-3.1 Å. Remarkably, each of the GSIs occupies the same general location on presenilin 1 (PS1) that accommodates the ß strand from amyloid precursor protein or Notch, interfering with substrate recruitment. L685,458 directly coordinates the two catalytic aspartate residues of PS1. E2012 binds to an allosteric site of γ-secretase on the extracellular side, potentially explaining its modulating activity. Structural analysis reveals a set of shared themes and variations for inhibitor and modulator recognition that will guide development of the next-generation substrate-selective inhibitors.