RESUMO
Networks of H-bonds can show non-additive behaviour, where the strength of one interaction perturbs another. The magnitude of such cooperative effects can be quantified by measuring the effect of the presence of an intramolecular H-bond at one site on a molecule on the association constant for formation of an intermolecular H-bond at another site. This approach has been used to quantify the cooperativity associated with the interaction of a primary amine with two H-bond acceptors. A series of compounds that have an intramolecular H-bond between an aniline NH2 group and a pyridine nitrogen were prepared, using polarising substituents on the pyridine ring to vary the strength of the intramolecular H-bond. The presence of the intramolecular interaction was confirmed by X-ray crystallography in the solid state and NMR spectroscopy in n-octane solution. UV-vis absorption titrations were used to measure the association constants for formation of an intermolecular H-bond with tri-n-butyl phosphine oxide in n-octane. Electron-donating substituents on the pyridine ring, which increase the strength of the intramolecular H-bond, were found to decrease the strength of the intermolecular H-bond between the aniline and the phosphine oxide. The results were used to determine the H-bond donor parameters for the anilines, α, and there is a linear relationship between the values of α and the H-bond acceptor parameter of the pyridine group involved in the intramolecular H-bond, ß. The slope of this relationship was used to determine the cooperativity parameter (κ = -0.10), which quantifies the negative allosteric cooperativity between the two H-bonding interactions. Calculated molecular electrostatic potential surfaces of the anilines quantitatively reproduce the experimental result, which suggests that effects are electrostatic in origin, either due to polarisation of the NH bonds or due to secondary electrostatic interactions between the two H-bond acceptors.
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Achieving oral bioavailability with Proteolysis Targeting Chimeras (PROTACs) is a key challenge. Here, we report the in vivo pharmacokinetic properties in mouse, rat, and dog of four clinical oral PROTACs and compare with an internally derived data set. We use NMR to determine 3D molecular conformations and structural preorganization free in solution, and we introduce the new experimental descriptors, solvent-exposed H-bond donors (eHBD), and acceptors (eHBA). We derive an upper limit of eHBD ≤ 2 for oral PROTACs in apolar environments and show a greater tolerance for other properties (eHBA, polarity, lipophilicity, and molecular weight) than for Rule-of-5 compliant oral drugs. Within a set of structurally related PROTACs, we show that examples with eHBD > 2 have much lower oral bioavailability than those that have eHBD ≤ 2. We summarize our findings as an experimental "Rule-of-oral-PROTACs" in order to assist medicinal chemists to achieve oral bioavailability in this challenging space.
Assuntos
Disponibilidade Biológica , Proteólise , Animais , Administração Oral , Cães , Camundongos , Ratos , Proteólise/efeitos dos fármacos , Humanos , Masculino , Ligação de Hidrogênio , Quimera de Direcionamento de ProteóliseRESUMO
Targeting the estrogen receptor alpha (ERα) pathway is validated in the clinic as an effective means to treat ER+ breast cancers. Here we present the development of a VHL-targeting and orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of ERα. In vitro studies with this PROTAC demonstrate excellent ERα degradation and ER antagonism in ER+ breast cancer cell lines. However, upon dosing the compound in vivo we observe an in vitro-in vivo disconnect. ERα degradation is lower in vivo than expected based on the in vitro data. Investigation into potential causes for the reduced maximal degradation reveals that metabolic instability of the PROTAC linker generates metabolites that compete for binding to ERα with the full PROTAC, limiting degradation. This observation highlights the requirement for metabolically stable PROTACs to ensure maximal efficacy and thus optimisation of the linker should be a key consideration when designing PROTACs.
Assuntos
Receptor alfa de Estrogênio , Proteólise , Proteína Supressora de Tumor Von Hippel-Lindau , Humanos , Receptor alfa de Estrogênio/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Feminino , Proteólise/efeitos dos fármacos , Animais , Administração Oral , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/administração & dosagemRESUMO
The structure-based design of small-molecule inhibitors targeting protein-protein interactions (PPIs) remains a huge challenge as the drug must bind typically wide and shallow protein sites. A PPI target of high interest for hematological cancer therapy is myeloid cell leukemia 1 (Mcl-1), a prosurvival guardian protein from the Bcl-2 family. Despite being previously considered undruggable, seven small-molecule Mcl-1 inhibitors have recently entered clinical trials. Here, we report the crystal structure of the clinical-stage inhibitor AMG-176 bound to Mcl-1 and analyze its interaction along with clinical inhibitors AZD5991 and S64315. Our X-ray data reveal high plasticity of Mcl-1 and a remarkable ligand-induced pocket deepening. Nuclear Magnetic Resonance (NMR)-based free ligand conformer analysis demonstrates that such unprecedented induced fit is uniquely achieved by designing highly rigid inhibitors, preorganized in their bioactive conformation. By elucidating key chemistry design principles, this work provides a roadmap for targeting the largely untapped PPI class more successfully.
Assuntos
Apoptose , Naftalenos , Modelos Moleculares , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , LigantesRESUMO
The determination of intracellular drug concentrations can provide a better understanding of the drug function and efficacy. Ideally, this should be performed nondestructively, with no modification of either the drug or the target, and with the capability to detect low amounts of the molecule of interest, in many cases in the µM to nM range (pmol to fmol per million cells). Unfortunately, it is currently challenging to have an experimental technique that provides direct quantitative measurements of intracellular drug concentrations that simultaneously satisfies these requirements. Here, we show that magic-angle spinning dynamic nuclear polarization (MAS DNP) can be used to fulfill these requirements. We apply a quantitative 15N MAS DNP approach in combination with 15N labeling to quantify the intracellular amount of the drug [15N]CHIR-98014, an activator of the Wingless and Int-1 signaling pathway, determining intracellular drug amounts in the range of tens to hundreds of picomoles per million cells. This is, to our knowledge, the first time that MAS DNP has been used to successfully estimate intracellular drug amounts.
Assuntos
Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodosRESUMO
Dynamic nuclear polarization (DNP) allows to dramatically enhance the sensitivity of magic angle spinning nuclear magnetic resonance (MAS NMR). DNP experiments usually rely on the detection of low-γ nuclei hyperpolarized from 1H with the use of cross polarization (CP), which assures more efficient signal enhancement. However, CP is usually not quantitative. Here we determine the quantification performance of three different approaches used in MAS NMR, (conventional CP, variable contact time CP, and multiple-contact CP) under DNP conditions, and we show that absolute quantification in MAS DNP NMR is possible, with errors below 10%.
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Pharmacological inhibition of cathepsin S (CatS) allows for a specific modulation of the adaptive immune system and many major diseases. Here, we used NMR fragment screening and crystal structure-aided merging to synthesize novel, highly selective CatS inhibitors with picomolar enzymatic Ki values and nanomolar functional activity in human Raji cells. Noncovalent fragment hits revealed binding hotspots, while the covalent inhibitor structure-activity relationship enabled efficient potency optimization.
Assuntos
Amidas/farmacologia , Inibidores Enzimáticos/farmacologia , Amidas/síntese química , Amidas/química , Catepsinas , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Genome-wide-association studies in chronic low back pain patients identified sepiapterin reductase as a high interest target for developing new analgesics. Here we used 19F NMR fragment screening for the discovery of novel, ligand-efficient SPR inhibitors. We report the crystal structures of six chemically diverse inhibitors complexed with SPR, identifying relevant interactions and binding modes in the sepiapterin pocket. Exploration of our initial fragment screening hit led to double-digit nanomolar inhibitors of SPR with excellent ligand efficiency.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Amidas/química , Inibidores Enzimáticos/química , Oxirredutases do Álcool/metabolismo , Amidas/síntese química , Amidas/metabolismo , Cristalografia por Raios X , Descoberta de Drogas , Estabilidade de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
PDZ (PSD-95, Dlg, ZO-1) domains are ubiquitous interaction modules that are involved in many cellular signal transduction pathways. Interference with PDZ-mediated protein-protein interactions has important implications in disease-related signaling processes. For this reason, PDZ domains have gained attention as potential targets for inhibitor design and, in the long run, drug development. Herein we report the development of small molecules to probe the function of the PDZ domain from human AF6 (ALL1-fused gene from chromosomeâ 6), which is an essential component of cell-cell junctions. These compounds bind to AF6 PDZ with substantially higher affinity than the peptide (Ile-Gln-Ser-Val-Glu-Val) derived from its natural ligand, EphB2. In intact cells, the compounds inhibit the AF6-Bcr interaction and interfere with epidermal growth factor (EGF)-dependent signaling.
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Cinesinas/antagonistas & inibidores , Miosinas/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Cinesinas/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Miosinas/metabolismo , Domínios PDZ , Peptídeos/química , Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptor EphB2/química , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-AtividadeRESUMO
The antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital for Mycobacterium tuberculosis due to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential for M. tuberculosis virulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition of M. tuberculosis division in broth culture. I3-AG85 also inhibited M. tuberculosis survival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistant M. tuberculosis strains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of the M. tuberculosis envelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of the M. tuberculosis envelope. I3-AG85 inhibited the division of and reduced TDM synthesis in an M. tuberculosis strain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design.
Assuntos
Aciltransferases/antagonistas & inibidores , Fatores Corda/antagonistas & inibidores , Fatores Corda/biossíntese , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Animais , Antígenos de Bactérias , Células da Medula Óssea/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura , Farmacorresistência Bacteriana , Farmacorresistência Bacteriana Múltipla , Feminino , Lipídeos/biossíntese , Macrófagos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Oxazinas , Proteínas Recombinantes/biossíntese , Tioglucosídeos/farmacologia , Uracila/metabolismo , XantenosRESUMO
With 1.6 million casualties annually and 2 billion people being infected, tuberculosis is still one of the most pressing healthcare challenges. Here we report on the new computational docking algorithm FRIGATE which unites continuous local optimization techniques (conjugate gradient method) with an inherently discrete computational approach in forcefield computation, resulting in equal or better scoring accuracies than several benchmark docking programs. By utilizing FRIGATE for a virtual screen of the ZINC library against the Mycobacterium tuberculosis (Mtb) enzyme antigen 85C, we identified novel small molecule inhibitors of multiple drug-resistant Mtb, which bind in vitro to the catalytic site of antigen 85C.
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Antituberculosos/farmacologia , Biologia Computacional/métodos , Mycobacterium tuberculosis/metabolismo , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Algoritmos , Antituberculosos/química , Proteínas de Bactérias/química , Sítios de Ligação , Domínio Catalítico , Química Farmacêutica/métodos , Desenho de Fármacos , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Reprodutibilidade dos Testes , Software , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
Synthetic peptides that specifically bind nuclear hormone receptors offer an alternative approach to small molecules for the modulation of receptor signaling and subsequent gene expression. Here we describe the design of a series of novel stapled peptides that bind the coactivator peptide site of estrogen receptors. Using a number of biophysical techniques, including crystal structure analysis of receptor-stapled peptide complexes, we describe in detail the molecular interactions and demonstrate that all-hydrocarbon staples modulate molecular recognition events. The findings have implications for the design of stapled peptides in general.
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Desenho de Fármacos , Peptídeos/síntese química , Receptores de Estrogênio/metabolismo , Cristalografia por Raios X , Humanos , Peptídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Receptores de Estrogênio/químicaRESUMO
Protein target-based discovery of novel antibiotics has been largely unsuccessful despite rich genome information. Particularly in need are new antibiotics for tuberculosis, which kills 1.6 million people annually and shows a rapid increase in multiple-drug-resistant cases. By combining fragment-based drug discovery with early whole cell antibacterial screening, we discovered novel ligand-efficient inhibitors of multiple-drug resistant Mycobacterium tuberculosis (Mtb), which bind to the substrate site of the Mtb protein antigen 85C, hitherto unused in Mtb chemotherapy.
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Aciltransferases/química , Antígenos de Bactérias/química , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Antituberculosos/química , Antituberculosos/imunologia , Domínio Catalítico , Resistência Microbiana a Medicamentos , Resistência a Múltiplos Medicamentos , Mycobacterium tuberculosis/imunologia , Ressonância Magnética Nuclear BiomolecularRESUMO
Nuclear magnetic resonance (NMR)-fragment-screening is a useful tool for lead identification and optimization in cases where shallow binding pockets have to be targeted, where highly selective lead structures are to be designed, and where novel, best-fit scaffolds for combinatorial library design are required. Robust and complementary protein- and ligand-detected NMR-screening methods are available for target proteins of various molecular weights and from different expression systems. The advantages of the NMR assay over other analytical techniques are high sensitivity toward loose fragment binders, comparatively high throughput and the ability to offer unique structural binding site information.
Assuntos
Desenho de Fármacos , Espectroscopia de Ressonância Magnética , Farmacologia/instrumentação , Ligantes , Modelos Moleculares , Ligação Proteica , Receptores de Droga/química , Receptores de Droga/metabolismoRESUMO
High-throughput screening of libraries containing compounds of 'drug-like' molecular weight has frequently resulted in no or poor drug candidates, especially when screening against demanding drug targets such as protein-protein interactions. Fragment-based lead discovery and optimization has evolved as a promising solution to this problem by combining the universal adaptability of low-molecular-weight fragments with immediate structural information on fragment binding modes. This review focuses on nuclear magnetic resonance (NMR) fragment screening techniques, which provide a unique combination of medium-throughput, direct binding site information and broad applicability. The utility and exemplary data of chemical shift-detected NMR fragment screening applied to the challenging protein-protein interaction target PDZ domains are summarized.
Assuntos
Desenho de Fármacos , Espectroscopia de Ressonância Magnética , Ligação Proteica/fisiologia , Animais , Técnicas de Química Combinatória , Humanos , Modelos Moleculares , Conformação Molecular , ProteômicaRESUMO
The N-terminal domain of the vaccinia virus protein E3L (Z alpha(E3L)) is essential for full viral pathogenicity in mice. It has sequence similarity to the high-affinity human Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). Here, we report the solution structure of Z alpha(E3L) and the chemical shift map of its interaction surface with Z-DNA. The global structure and the Z-DNA interaction surface of Z alpha(E3L) are very similar to the high-affinity Z-DNA-binding domains Z alpha(ADAR1) and Z alpha(DLM1). However, the key Z-DNA contacting residue Y48 of Z alpha(E3L) adopts a different side chain conformation in unbound Z alpha(E3L), which requires rearrangement for binding to Z-DNA. This difference suggests a molecular basis for the significantly lower in vitro affinity of Z alpha(E3L) to Z-DNA compared with its homologues.