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1.
Biochemistry ; 62(22): 3206-3213, 2023 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-37938120

RESUMO

KRAS GTPases harbor oncogenic mutations in more than 25% of human tumors. KRAS is considered to be largely undruggable due to the lack of a suitable small-molecule binding site. Here, we report a unique crystal structure of His-tagged KRASG12D that reveals a remarkable conformational change. The Switch I loop of one His-KRASG12D structure extends into the Switch I/II pocket of another His-KRASG12D in an adjacent unit cell to create an elaborate interface that is reminiscent of high-affinity protein-protein complexes. We explore the contributions of amino acids at this interface using alanine-scanning studies with alchemical free energy perturbation calculations based on explicit-solvent molecular dynamics simulations. Several interface amino acids were found to be hot spots as they contributed more than 1.5 kcal/mol to the protein-protein interaction. Computational analysis of the complex revealed the presence of two large binding pockets that possess physicochemical features typically found in pockets considered druggable. Small-molecule binding to these pockets may stabilize this autoinhibited structure of KRAS if it exists in cells to provide a new strategy to inhibit RAS signaling.


Assuntos
Proteínas Proto-Oncogênicas p21(ras) , Transdução de Sinais , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Simulação de Dinâmica Molecular , Ligação Proteica , Aminoácidos , Mutação
2.
Proc Natl Acad Sci U S A ; 117(13): 7131-7139, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32179690

RESUMO

Ral (Ras-like) GTPases are directly activated by oncogenic Ras GTPases. Mutant K-Ras (G12C) has enabled the development of covalent K-Ras inhibitors currently in clinical trials. However, Ral, and the overwhelming majority of mutant oncogenic K-Ras, are devoid of a druggable pocket and lack an accessible cysteine for the development of a covalent inhibitor. Here, we report that covalent bond formation by an aryl sulfonyl fluoride electrophile at a tyrosine residue (Tyr-82) inhibits guanine exchange factor Rgl2-mediated nucleotide exchange of Ral GTPase. A high-resolution 1.18-Å X-ray cocrystal structure shows that the compound binds to a well-defined binding site in RalA as a result of a switch II loop conformational change. The structure, along with additional high-resolution crystal structures of several analogs in complex with RalA, confirm the importance of key hydrogen bond anchors between compound sulfone oxygen atoms and Ral backbone nitrogen atoms. Our discovery of a pocket with features found on known druggable sites and covalent modification of a bystander tyrosine residue present in Ral and Ras GTPases provide a strategy that could lead to therapeutic agent targeting oncogenic Ras mutants that are devoid of a cysteine nucleophile.


Assuntos
Proteínas ral de Ligação ao GTP/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Bibliotecas de Moléculas Pequenas , Proteínas ral de Ligação ao GTP/metabolismo
3.
Proc Natl Acad Sci U S A ; 115(45): E10566-E10575, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30355767

RESUMO

Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1 subunit (CaVα1) that is engaged in protein-protein interactions with auxiliary α2/δ and ß subunits. The high-affinity CaV2.2α1⋅CaVß3 protein-protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1⋅CaVß3 protein-protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1 Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVß3 Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein-protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacocinética , Feminino , Células HEK293 , Humanos , Transporte de Íons , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Camundongos , Neuralgia/prevenção & controle , Nociceptividade/efeitos dos fármacos , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Bibliotecas de Moléculas Pequenas/farmacocinética
4.
Nature ; 515(7527): 443-7, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25219851

RESUMO

The Ras-like GTPases RalA and RalB are important drivers of tumour growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here we used protein structure analysis and virtual screening to identify drug-like molecules that bind to a site on the GDP-bound form of Ral. The compounds RBC6, RBC8 and RBC10 inhibited the binding of Ral to its effector RALBP1, as well as inhibiting Ral-mediated cell spreading of murine embryonic fibroblasts and anchorage-independent growth of human cancer cell lines. The binding of the RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasmon resonance and (1)H-(15)N transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy. RBC8 and BQU57 show selectivity for Ral relative to the GTPases Ras and RhoA and inhibit tumour xenograft growth to a similar extent to the depletion of Ral using RNA interference. Our results show the utility of structure-based discovery for the development of therapeutics for Ral-dependent cancers.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais , Terapia de Alvo Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas ral de Ligação ao GTP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Simulação por Computador , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ral de Ligação ao GTP/química , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo
5.
Bioorg Med Chem ; 26(23-24): 6128-6134, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30470597

RESUMO

Bone is a common site of metastasis for breast, prostate, lung, kidney and other cancers. Bone metastases are incurable, and substantially reduce patient quality of life. To date, there exists no small-molecule therapeutic agent that can reduce tumor burden in bone. This is partly attributed to the lack of suitable in vitro assays that are good models of tumor growth in bone. Here, we take advantage of a novel ex vivo model of bone colonization to report a series of pyrrolopyrazolone small molecules that inhibit cancer cell invasion and ex vivo tumor growth in bone at single-digit micromolar concentration. We find that the compounds modulated the expression levels of genes associated with bone-forming osteoblasts, bone-destroying osteoclasts, cancer cell viability and metastasis. Our compounds provide chemical tools to uncover novel targets and pathways associated with bone metastasis, as well as for the development of compounds to prevent and reverse bone tumor growth in vivo.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Estrutura Molecular , Gravidez , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
6.
Biochemistry ; 56(12): 1768-1784, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28186725

RESUMO

Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.


Assuntos
Pirazóis/síntese química , Pirróis/síntese química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/síntese química , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Cristalografia por Raios X , Corantes Fluorescentes/química , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica/efeitos dos fármacos , Pirazóis/farmacologia , Pirróis/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Coloração e Rotulagem/métodos , Relação Estrutura-Atividade , Termodinâmica , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
7.
J Chem Inf Model ; 57(9): 2250-2272, 2017 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-28766941

RESUMO

The binding affinity of a protein-protein interaction is concentrated at amino acids known as hot spots. It has been suggested that small molecules disrupt protein-protein interactions by either (i) engaging receptor protein hot spots or (ii) mimicking hot spots of the protein ligand. Yet, no systematic studies have been done to explore how effectively existing small-molecule protein-protein interaction inhibitors mimic or engage hot spots at protein interfaces. Here, we employ explicit-solvent molecular dynamics simulations and end-point MM-GBSA free energy calculations to explore this question. We select 36 compounds for which high-quality binding affinity and cocrystal structures are available. Five complexes that belong to three classes of protein-protein interactions (primary, secondary, and tertiary) were considered, namely, BRD4•H4, XIAP•Smac, MDM2•p53, Bcl-xL•Bak, and IL-2•IL-2Rα. Computational alanine scanning using MM-GBSA identified hot-spot residues at the interface of these protein interactions. Decomposition energies compared the interaction of small molecules with individual receptor hot spots to those of the native protein ligand. Pharmacophore analysis was used to investigate how effectively small molecules mimic the position of hot spots of the protein ligand. Finally, we study whether small molecules mimic the effects of the native protein ligand on the receptor dynamics. Our results show that, in general, existing small-molecule inhibitors of protein-protein interactions do not optimally mimic protein-ligand hot spots, nor do they effectively engage protein receptor hot spots. The more effective use of hot spots in future drug design efforts may result in smaller compounds with higher ligand efficiencies that may lead to greater success in clinical trials.


Assuntos
Simulação de Dinâmica Molecular , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Cristalografia por Raios X , Ligantes , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Termodinâmica
8.
Bioorg Med Chem ; 25(12): 2995-3005, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28438385

RESUMO

Triple-negative breast cancers (TNBCs) lack the signature targets of other breast tumors, such as HER2, estrogen receptor, and progesterone receptor. These aggressive basal-like tumors are driven by a complex array of signaling pathways that are activated by multiple driver mutations. Here we report the discovery of 6 (KIN-281), a small molecule that inhibits multiple kinases including maternal leucine zipper kinase (MELK) and the non-receptor tyrosine kinase bone marrow X-linked (BMX) with single-digit micromolar IC50s. Several derivatives of 6 were synthesized to gain insight into the binding mode of the compound to the ATP binding pocket. Compound 6 was tested for its effect on anchorage-dependent and independent growth of MDA-MB-231 and MDA-MB-468 breast cancer cells. The effect of 6 on BMX prompted us to evaluate its effect on STAT3 phosphorylation and DNA binding. The compound's inhibition of cell growth led to measurements of survivin, Bcl-XL, p21WAF1/CIP1, and cyclin A2 levels. Finally, LC3B-II levels were quantified following treatment of cells with 6 to determine whether the compound affected autophagy, a process that is known to be activated by STAT3. Compound 6 provides a starting point for the development of small molecules with polypharmacology that can suppress TNBC growth and metastasis.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Autofagia/efeitos dos fármacos , Mama/efeitos dos fármacos , Mama/metabolismo , Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Simulação de Acoplamento Molecular , Fator de Transcrição STAT3/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/metabolismo
9.
J Chem Inf Model ; 56(6): 1139-51, 2016 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-27154487

RESUMO

Virtual screening consists of docking libraries of small molecules to a target protein followed by rank-ordering of the resulting structures using scoring functions. The ability of scoring methods to distinguish between actives and inactives depends on several factors that include the accuracy of the binding pose during the docking step and the quality of the three-dimensional structure of the target. Here, we build on our previous work to introduce a new scoring approach (SVMGen) that uses machine learning trained with features from statistical pair potentials obtained from three-dimensional crystal structures. We use SVMGen and GlideScore to explore how enrichment or rank-ordering is affected by binding pose accuracy. To that end, we create a validation set that consists strictly of proteins whose crystal structure was solved in complex with their inhibitors. For the rank-ordering studies, we use crystal structures from PDBbind along with corresponding binding affinity data provided in the database. In addition to binding pose, we investigate the effect of using modeled structures for the target on the enrichment performance of SVMGen and GlideScore. To accomplish this, we generated homology models for protein kinases in DUD-E for which crystal structures are available to enable comparison of enrichment between modeled and crystal structure. We also generate homology models for kinases in SARfari for which there are many known small-molecule inhibitors but no known crystal structure. These models are used to assess the ability of SVMGen and GlideScore to distinguish between actives and decoys. We focus our work on protein kinases considering the wealth of structural and binding affinity data that exists for this family of proteins.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/química , Máquina de Vetores de Suporte , Cristalografia por Raios X , Ligantes , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Interface Usuário-Computador
10.
J Chem Inf Model ; 54(7): 2105-16, 2014 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-24856086

RESUMO

Molecular recognition is a complex process that involves a large ensemble of structures of the receptor and ligand. Yet, most structure-based virtual screening is carried out on a single structure typically from X-ray crystallography. Explicit-solvent molecular dynamics (MD) simulations offer an opportunity to sample multiple conformational states of a protein. Here we evaluate our recently developed scoring method SVMSP in its ability to enrich chemical libraries docked to MD structures of seven proteins from the Directory of Useful Decoys (DUD). SVMSP is a target-specific rescoring method that combines machine learning with statistical potentials. We find that enrichment power as measured by the area under the ROC curve (ROC-AUC) is not affected by increasing the number of MD structures. Among individual MD snapshots, many exhibited enrichment that was significantly better than the crystal structure, but no correlation between enrichment and structural deviation from crystal structure was found. We followed an innovative approach by training SVMSP scoring models using MD structures (SVMSPMD). The resulting models were applied to two difficult cases (p38 and CDK2) for which enrichment was not better than random. We found remarkable increase in enrichment power, particularly for p38, where the ROC-AUC increased by 0.30 to 0.85. Finally, we explored approaches for a priori identification of MD snapshots with high enrichment power from an MD simulation in the absence of active compounds. We found that the use of randomly selected compounds docked to the target of interest using SVMSP led to notable enrichment for EGFR and Src MD snapshots. SVMSP rescoring of protein-compound MD structures was applied for the search of small-molecule inhibitors of the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2). Rank-ordering of a commercial library of 50 000 compounds docked to MD structures of ALDH2 led to five small-molecule inhibitors. Four compounds had IC50s below 5 µM. These compounds serve as leads for the design and synthesis of more potent and selective ALDH2 inhibitors.


Assuntos
Aldeído Desidrogenase/química , Aldeído Desidrogenase/metabolismo , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/metabolismo , Aldeído Desidrogenase/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Conformação Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Máquina de Vetores de Suporte
11.
J Chem Inf Model ; 53(10): 2659-70, 2013 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-24032517

RESUMO

End-point free energy calculations using MM-GBSA and MM-PBSA provide a detailed understanding of molecular recognition in protein-ligand interactions. The binding free energy can be used to rank-order protein-ligand structures in virtual screening for compound or target identification. Here, we carry out free energy calculations for a diverse set of 11 proteins bound to 14 small molecules using extensive explicit-solvent MD simulations. The structure of these complexes was previously solved by crystallography and their binding studied with isothermal titration calorimetry (ITC) data enabling direct comparison to the MM-GBSA and MM-PBSA calculations. Four MM-GBSA and three MM-PBSA calculations reproduced the ITC free energy within 1 kcal·mol(-1) highlighting the challenges in reproducing the absolute free energy from end-point free energy calculations. MM-GBSA exhibited better rank-ordering with a Spearman ρ of 0.68 compared to 0.40 for MM-PBSA with dielectric constant (ε = 1). An increase in ε resulted in significantly better rank-ordering for MM-PBSA (ρ = 0.91 for ε = 10), but larger ε significantly reduced the contributions of electrostatics, suggesting that the improvement is due to the nonpolar and entropy components, rather than a better representation of the electrostatics. The SVRKB scoring function applied to MD snapshots resulted in excellent rank-ordering (ρ = 0.81). Calculations of the configurational entropy using normal-mode analysis led to free energies that correlated significantly better to the ITC free energy than the MD-based quasi-harmonic approach, but the computed entropies showed no correlation with the ITC entropy. When the adaptation energy is taken into consideration by running separate simulations for complex, apo, and ligand (MM-PBSAADAPT), there is less agreement with the ITC data for the individual free energies, but remarkably good rank-ordering is observed (ρ = 0.89). Interestingly, filtering MD snapshots by prescoring protein-ligand complexes with a machine learning-based approach (SVMSP) resulted in a significant improvement in the MM-PBSA results (ε = 1) from ρ = 0.40 to ρ = 0.81. Finally, the nonpolar components of MM-GBSA and MM-PBSA, but not the electrostatic components, showed strong correlation to the ITC free energy; the computed entropies did not correlate with the ITC entropy.


Assuntos
Simulação de Dinâmica Molecular , Proteínas/química , Proteoma/química , Bibliotecas de Moléculas Pequenas/química , Animais , Sítios de Ligação , Calorimetria , Bases de Dados de Proteínas , Descoberta de Drogas , Humanos , Cinética , Ligantes , Ligação Proteica , Conformação Proteica , Proteínas/agonistas , Proteínas/antagonistas & inibidores , Termodinâmica , Interface Usuário-Computador
12.
Bioorg Med Chem ; 21(7): 2145-55, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23411397

RESUMO

The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 µM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. Compound 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5h and peak concentration of 5 µM. Similar levels of the inhibitor were detected in tumor tissue up to 10h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Metástase Neoplásica/tratamento farmacológico , Mapas de Interação de Proteínas/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/uso terapêutico , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Mama/efeitos dos fármacos , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
13.
ACS Chem Neurosci ; 14(14): 2509-2516, 2023 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-37382289

RESUMO

Ras homolog gene family member (RhoA) is a GTPase and a member of the RAS superfamily of GTPases. RhoA is a master regulator of the actin cytoskeleton. It inhibits axon growth preventing repair and recovery following spinal cord and traumatic brain injuries. Despite decades of research into the biological function of Rho GTPases, there exist no small-molecule Rho inhibitors. Here, we screen a library of cysteine electrophiles to explore whether covalent bond formation at Cys-107 leads to inhibition of RhoA activation by guanine exchange factor Trio. Two fragments, propiolamide 1 (ACR-895) and acrylamide 2 (ACR-917), inhibited RhoA nucleotide exchange by Trio in a time-dependent manner. The fragments formed a covalent bond with wild-type RhoA but not Cys107Ser RhoA mutant. Time- and concentration-dependent studies led to equilibrium constants KIs and reaction rates that correspond to t1/2 values in the single-digit hour range. One fragment was selective for RhoA over Rac1 GTPase and had no effect on KRAS nucleotide exchange by SOS1. The fragments did not inhibit RhoA binding to ROCK effector protein. This work establishes Cys-107 as a suitable site for Rho GTPase inhibition and provides fragment starting points for the future development of Rho GTPase covalent inhibitors that could have profound implications in the treatment of patients with injuries of the central nervous system.


Assuntos
Fatores de Troca do Nucleotídeo Guanina , Guanina , Humanos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Nucleotídeos/metabolismo
14.
RSC Med Chem ; 14(9): 1803-1816, 2023 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-37731696

RESUMO

Transcriptional enhanced associate domain (TEAD) binding to co-activator yes-associated protein (YAP1) leads to a transcription factor of the Hippo pathway. TEADs are regulated by S-palmitoylation of a conserved cysteine located in a deep well-defined hydrophobic pocket outside the TEAD·YAP1 interaction interface. Previously, we reported the discovery of a small molecule based on the structure of flufenamic acid that binds to the palmitate pocket, forms a covalent bond with the conserved cysteine, and inhibits TEAD4 binding to YAP1. Here, we screen a fragment library of chloroacetamide electrophiles to identify new scaffolds that bind to the palmitate pocket of TEADs and disrupt their interaction with YAP1. Time- and concentration-dependent studies with wild-type and mutant TEAD1-4 provided insight into their reaction rates and binding constants and established the compounds as covalent inhibitors of TEAD binding to YAP1. Binding pose hypotheses were generated by covalent docking revealing that the fragments and compounds engage lower, middle, and upper sub-sites of the palmitate pocket. Our fragments and compounds provide new scaffolds and starting points for the design of derivatives with improved inhibition potency of TEAD palmitoylation and binding to YAP1.

15.
J Med Chem ; 66(1): 266-284, 2023 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-36562717

RESUMO

Transcriptional enhanced associate domains (TEADs) are transcription factors that bind to cotranscriptional activators like the yes-associated protein (YAP) or its paralog transcriptional coactivator with a PDZ-binding motif (TAZ). TEAD·YAP/TAZ target genes are involved in tissue and immune homeostasis, organ size control, tumor growth, and metastasis. Here, we report isoindoline and octahydroisoindole small molecules with a cyanamide electrophile that forms a covalent bond with a conserved cysteine in the TEAD palmitate-binding cavity. Time- and concentration-dependent studies against TEAD1-4 yielded second-order rate constants kinact/KI greater than 100 M-1 s-1. Compounds inhibited YAP1 binding to TEADs with submicromolar IC50 values. Cocrystal structures with TEAD2 enabled structure-activity relationship studies. In mammalian cells, compounds suppressed CTGF mRNA levels and inhibited TEAD1-4 transcriptional activity with submicromolar IC50 values. Inhibition of TEAD binding to YAP1 in mammalian cells was also observed. Several compounds inhibited the cell viability of sarcoma, hepatocellular carcinoma, glioblastoma, and breast cancer cells with single-digit micromolar IC50 values.


Assuntos
Cianamida , Neoplasias , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Sinalização YAP , Fatores de Transcrição/metabolismo , Mamíferos/metabolismo
16.
ChemMedChem ; 18(16): e202300272, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37269475

RESUMO

Ral RAS GTPases are directly activated by KRAS through a trimeric complex with a guanine exchange factor. Ral is considered undruggable and lacks an accessible cysteine for covalent drug development. Previously we had reported an aryl sulfonyl fluoride fragment that formed a covalent bond at Tyr-82 on Ral and created a deep and well-defined pocket. Here, we explore this pocket further through design and synthesis of several fragment derivatives. The fragment core is modified by introducing tetrahydronaphthalene or benzodioxane rings to enhance affinity and stability of the sulfonyl fluoride reactive group. The deep pocket in the Switch II region is also explored by modifying the aromatic ring of the fragment that is ensconced into the pocket. Compounds 19 (SOF-658) and 26 (SOF-648) formed a single robust adduct specifically at Tyr-82, inhibited Ral GTPase exchange in buffer and in mammalian cells, and blocked invasion of pancreatic ductal adenocarcinoma cancer cells. Compound 19 (SOF-658) was stable in buffer, mouse, and human microsomes suggesting that further optimization could lead to small molecules to probe Ral activity in tumor models.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Fatores de Troca do Nucleotídeo Guanina , Neoplasias Pancreáticas/patologia , GTP Fosfo-Hidrolases , Mamíferos
17.
Mol Pain ; 8: 54, 2012 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-22828369

RESUMO

BACKGROUND: The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al. Nature Medicine 17:822-829 (2011)]. RESULTS AND DISCUSSION: Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca²âº channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca²âº channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release compared to vehicle control. CONCLUSIONS: Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target.


Assuntos
Síndrome da Imunodeficiência Adquirida/terapia , Canais de Cálcio Tipo N/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Proteínas do Tecido Nervoso/química , Nociceptividade , Nociceptores/metabolismo , Peptídeos/uso terapêutico , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Síndrome da Imunodeficiência Adquirida/complicações , Sequência de Aminoácidos , Animais , Separação Celular , Modelos Animais de Doenças , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Hiperalgesia/complicações , Hiperalgesia/tratamento farmacológico , Hiperalgesia/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Neurotransmissores/metabolismo , Nociceptividade/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Nociceptores/patologia , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Doenças do Sistema Nervoso Periférico/etiologia , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
18.
Bioorg Med Chem ; 20(15): 4760-73, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22771232

RESUMO

The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.


Assuntos
Antineoplásicos/farmacologia , Benzoatos/farmacologia , Desenho de Fármacos , Lectinas de Ligação a Manose/antagonistas & inibidores , Glicoproteínas de Membrana/antagonistas & inibidores , Simulação de Dinâmica Molecular , Piperidinas/farmacologia , Receptores de Superfície Celular/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Benzoatos/síntese química , Benzoatos/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lectinas de Ligação a Manose/isolamento & purificação , Lectinas de Ligação a Manose/metabolismo , Glicoproteínas de Membrana/isolamento & purificação , Glicoproteínas de Membrana/metabolismo , Estrutura Molecular , Peso Molecular , Piperidinas/síntese química , Piperidinas/química , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Relação Estrutura-Atividade
19.
Nucleic Acids Res ; 38(Database issue): D765-73, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19923229

RESUMO

BioDrugScreen is a resource for ranking molecules docked against a large number of targets in the human proteome. Nearly 1600 molecules from the freely available NCI diversity set were docked onto 1926 cavities identified on 1589 human targets resulting in >3 million receptor-ligand complexes requiring >200,000 cpu-hours on the TeraGrid. The targets in BioDrugScreen originated from Human Cancer Protein Interaction Network, which we have updated, as well as the Human Druggable Proteome, which we have created for the purpose of this effort. This makes the BioDrugScreen resource highly valuable in drug discovery. The receptor-ligand complexes within the database can be ranked using standard and well-established scoring functions like AutoDock, DockScore, ChemScore, X-Score, GoldScore, DFIRE and PMF. In addition, we have scored the complexes with more intensive GBSA and PBSA approaches requiring an additional 120,000 cpu-hours on the TeraGrid. We constructed a simple interface to enable users to view top-ranking molecules and access purchasing and other information for further experimental exploration.


Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Bases de Dados de Ácidos Nucleicos , Desenho de Fármacos , Preparações Farmacêuticas/química , Proteoma , Proteômica/métodos , Biologia Computacional/tendências , Avaliação Pré-Clínica de Medicamentos/instrumentação , Humanos , Armazenamento e Recuperação da Informação/métodos , Internet , Ligantes , Ligação Proteica , Software
20.
ChemMedChem ; 17(6): e202100750, 2022 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-35061330

RESUMO

Ral GTPases belong to the RAS superfamily, and they are directly activated by K-RAS. The RalGEF pathway is one of the three major K-RAS signaling pathways. Ral GTPases do not possess a cysteine nucleophile to develop a covalent inhibitor following the strategy that led to a K-RAS G12C therapeutic agent. However, several cysteine amino acids exist on the surface of guanine exchange factors that activate Ral GTPases, such as Rgl2. Here, we screen a library of cysteine electrophile fragments to determine if covalent bond formation at one of the Rgl2 surface cysteines could inhibit Ral GTPase activation. We found several chloroacetamide and acrylamide fragments that inhibited Ral GTPase exchange by Rgl2. Site-directed mutagenesis showed that covalent bond formation at Cys-284, but not other cysteines, leads to inhibition of Ral activation by Rgl2. Follow-up time- and concentration-dependent studies of derivatives identified by substructure search of commercial libraries further confirmed Cys-284 as the reaction site and identified the indoline fragments as the most promising series for further development. Cys-284 is located outside of the Ral ⋅ Rgl2 interface on a loop that has several residues that come in direct contact with Ral GTPases. Our allosteric covalent fragment inhibitors provide a starting point for the development of small-molecule covalent inhibitors to probe Ral GTPases in animal models.


Assuntos
Cisteína , Fatores de Troca do Nucleotídeo Guanina , Animais , Sítios de Ligação , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transdução de Sinais
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