ABSTRACT
Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1-5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7-9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV.
Subject(s)
Anti-HIV Agents/pharmacology , Anti-HIV Agents/therapeutic use , Capsid Proteins/antagonists & inhibitors , HIV-1/drug effects , Adolescent , Adult , Anti-HIV Agents/chemistry , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Line , Cells, Cultured , Drug Resistance, Viral/genetics , Female , HIV-1/growth & development , Humans , Male , Middle Aged , Models, Molecular , Virus Replication/drug effects , Young AdultABSTRACT
Lenacapavir (LEN), a long-acting injectable, is the first approved human immunodeficiency virus type 1 capsid inhibitor and one of a few FDA-approved drugs that exhibit atropisomerism. LEN exists as a mixture of two class 2 atropisomers that interconvert at a fast rate (t1/2 <2 hours) with a ratio that is stable over time and unaffected by enzymes or binding to proteins in plasma. LEN exhibits low systemic clearance (CL) in nonclinical species and humans; however, in all species the observed CL was higher than the in vitro predicted CL. The volume of distribution was moderate in nonclinical species and consistent with the tissue distribution observed by whole body autoradiography in rats. LEN does not distribute to brain, consistent with being a P-glycoprotein (P-gp) substrate. Mechanistic drug disposition studies with [14C]LEN in IV-dosed BDC rats and dogs showed a substantial amount of unchanged LEN (31 - 60% of dose) excreted in feces, indicating that intestinal excretion (IE) was a major clearance pathway for LEN in both species. Coadministration of oral elacridar, a P-gp inhibitor, in rats decreased CL and IE of LEN. Renal excretion was <1% of dose in both species. In plasma, almost all radioactivity was unchanged LEN. Low levels of metabolites in excreta included LEN-conjugates with glutathione, pentose, and glucuronic acid, which were consistent with metabolites formed in vitro in Hµrel® hepatocyte cocultures and those observed in human. Our studies highlight the importance of IE for efflux substrates that are highly metabolically stable compounds with slow elimination rates. Significance Statement LEN is a long-acting injectable that exists as conformationally stable atropisomers. Due to an atropisomeric interconversion rate that significantly exceeds the in vivo elimination rate, the atropisomer ratio of LEN remains constant in circulation. The disposition of LEN highlights that intestinal excretion has a substantial part in the elimination of compounds that are metabolically highly stable and efflux transporter substrates.
ABSTRACT
Lenacapavir (LEN) is a picomolar first-in-class capsid inhibitor of human immunodeficiency virus type 1 (HIV-1) with a multistage mechanism of action and no known cross resistance to other existing antiretroviral (ARV) drug classes. LEN exhibits a low aqueous solubility and exceptionally low systemic clearance following intravenous (IV) administration in nonclinical species and humans. LEN formulated in an aqueous suspension or a PEG/water solution formulation showed sustained plasma exposure levels with no unintended rapid drug release following subcutaneous (SC) administration to rats and dogs. A high total fraction dose release was observed with both formulations. The long-acting pharmacokinetics (PK) were recapitulated in humans following SC administration of both formulations. The SC PK profiles displayed two-phase absorption kinetics in both animals and humans with an initial fast-release absorption phase, followed by a slow-release absorption phase. Noncompartmental and compartmental analyses informed the LEN systemic input rate from the SC depot and exit rate from the body. Modeling-enabled deconvolution of the input rates from two processes: absorption of the soluble fraction (minor) from a direct fast-release process leading to the early PK phase and absorption of the precipitated fraction (major) from an indirect slow-release process leading to the later PK phase. LEN SC PK showed flip-flop kinetics due to the input rate being substantially slower than the systemic exit rate. LEN input rates via the slow-release process in humans were slower than those in both rats and dogs. Overall, the combination of high potency, exceptional stability, and optimal release rate from the injection depot make LEN well suited for a parenteral long-acting formulation that can be administered once up to every 6 months in humans for the prevention and treatment of HIV-1.
Subject(s)
Anti-HIV Agents , HIV-1 , Humans , Rats , Animals , Dogs , Anti-Retroviral Agents , Capsid , Anti-HIV Agents/pharmacology , Capsid ProteinsABSTRACT
People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options1-3. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently4. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection.
Subject(s)
Anti-HIV Agents/pharmacology , Capsid Proteins/antagonists & inhibitors , HIV Infections/drug therapy , HIV-1/drug effects , Indazoles/pharmacology , Pyridines/pharmacology , Small Molecule Libraries/pharmacology , Animals , Anti-HIV Agents/therapeutic use , Capsid/drug effects , Capsid/metabolism , Capsid Proteins/genetics , DNA, Viral/drug effects , Delayed-Action Preparations , Drug Resistance, Viral/drug effects , HIV Infections/genetics , HIV Infections/virology , HIV-1/genetics , HIV-1/pathogenicity , HIV-2/drug effects , HIV-2/pathogenicity , Humans , Indazoles/therapeutic use , Medication Adherence , Mice , Pyridines/therapeutic useABSTRACT
Biologically active, therapeutically useful, DNA binding natural products continue to reveal new paradigms for sequence-selective recognition, to enlist beautiful mechanisms of in situ activation for DNA modification, to define new therapeutic targets, to exploit new mechanisms to achieve cellular selectivity, and to provide a rich source of new drugs. These attributes arise in compact structures of complex integrated function.
Subject(s)
Antineoplastic Agents/metabolism , DNA/metabolism , Animals , Antineoplastic Agents/pharmacology , DNA/chemistry , DNA/drug effects , Humans , Models, Molecular , Molecular Conformation , Protein Binding/drug effects , Stereoisomerism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
A protocol for a fluorescent intercalator displacement (FID) assay useful for establishing DNA binding selectivity, affinity, stoichiometry, and binding site size, and for distinguishing modes of DNA binding is presented.
Subject(s)
DNA/chemistry , DNA/metabolism , Fluorescent Dyes/chemistry , Intercalating Agents/chemistry , Spectrometry, Fluorescence/methods , Binding Sites , Ethidium/chemistry , Ethidium/metabolism , Fluorescent Dyes/metabolism , Intercalating Agents/metabolism , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolismABSTRACT
A summary of the qualitative and quantitative elements of a fluorescent intercalator displacement (FID) assay useful for establishing the DNA binding selectivity, affinity, stoichiometry, and binding site size and distinguishing modes of DNA binding is provided.
Subject(s)
DNA/chemistry , Fluorescent Dyes/chemistry , Spectrometry, Fluorescence/methods , Base Sequence , Binding, Competitive , DNA/metabolism , Fluorescent Dyes/metabolism , Netropsin/chemistry , Netropsin/metabolism , Nylons/chemistry , Nylons/metabolism , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Thiazoles/chemistry , Thiazoles/metabolismABSTRACT
Four hairpin polyamides bearing subtle N- and C-terminal substitutions were examined in a fluorescent intercalator displacement (FID) assay enlisting a library of 512 DNA hairpins that contain all possible five base pair sequences in a challenging probe of its capabilities for establishing DNA binding sequence selectivity. Not only did the assay define the global sequence selectivity expected based on known structural interactions and Dervan's pairing rules establishing the utility of the method for characterizing such polyamides, but previously unappreciated subtle substituent effects on global sequence selectivity were also revealed. Thus, we report the discovery of a novel five base pair high affinity binding site of the form 5'-WWCWW (vs 5'-WGWWW) for the polyamide ImPyPy-gamma-PyPyPy-beta-Dp and its structural basis.
Subject(s)
Nylons/chemical synthesis , Oligonucleotides/chemistry , Base Sequence , Binding Sites , DNA/chemistry , Fluorescent Dyes , Intercalating Agents , Nucleic Acid Conformation , Nylons/chemistry , Protein Structure, Secondary , Structure-Activity RelationshipABSTRACT
Protein titration displacement of ethidium bromide bound to hairpin deoxyoligonucleotides containing any sequence of interest provides a well-defined titration curve (measuring the loss of fluorescence derived from the DNA bound ethidium bromide) that provides both absolute binding constants (K(a)) and stoichiometry of binding. This use of a fluorescent intercalator displacement (FID) assay for establishing protein DNA binding affinity and selectivity is demonstrated with the examination of the LEF-1 HMG domain binding to hairpin deoxyoligonucleotides containing its commonly accepted consensus sequence 5'-CTTTGWW (W=A or T) and those modified (5'-CTNTGWW) to examine sequences implicated in early studies (5'-CTNTG). The effectiveness of the FID assay coupled with its technically non-demanding experimental use makes it an attractive alternative or complement to selection screening, footprinting or affinity cleavage, and electrophoretic mobility shift assays for detecting, characterizing, and quantitating protein DNA binding affinity and selectivity.
Subject(s)
DNA/chemistry , Fluorescent Dyes/chemistry , Intercalating Agents/chemistry , Proteins/chemistry , Animals , Chemical Phenomena , Chemistry, Physical , DNA-Binding Proteins/pharmacology , Ethidium , Lymphoid Enhancer-Binding Factor 1 , Mice , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Transcription Factors/pharmacologyABSTRACT
The binding affinities of several triplex forming oligonucleotides were determined using a fluorescent intercalator displacement (FID) assay.