RESUMEN
Aging workers of the termite Neocapritermes taracua can defend their colony by sacrificing themselves by body rupture, mixing the externally stored blue laccase BP76 with hydroquinones to produce a sticky liquid rich in toxic benzoquinones. Here, we describe the crystal structure of BP76 isolated from N. taracua in its native form. The structure reveals several stabilization strategies, including compact folding, glycosylation, and flexible loops with disulfide bridges and tight dimer interface. The remarkable stability of BP76 maintains its catalytic activity in solid state during the lifespan of N. taracua workers, providing old workers with an efficient defensive weapon to protect their colony.
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Reactive N-hydroxy-9-azabicyclo[3.3.1]nonane (ABNOH) linked 2'-deoxyuridine 5'-O-mono- and triphosphates were synthesized through a CuAAC reaction of ABNOH-PEG4-N3 with 5-ethynyl-dUMP or -dUTP. The modified triphosphate was used as substrate for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The keto-ABNO radical reacted with tryptophan (Trp) and Trp-containing peptides to form a stable tricyclic fused hexahydropyrrolo-indole conjugates. Similarly modified ABNOH-linked nucleotides reacted with Trp-containing peptides to form a stable conjugate in the presence but surprisingly even in the absence of NaNO2 (presumably through activation by O2). The reactive ABNOH-modified DNA probe was used for bioconjugations and crosslinking with Trp-containing peptides or proteins.
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The SorC family of transcriptional regulators plays a crucial role in controlling the carbohydrate metabolism and quorum sensing. We employed an integrative approach combining X-ray crystallography and cryo-electron microscopy to investigate architecture and functional mechanism of two prototypical representatives of two sub-classes of the SorC family: DeoR and CggR from Bacillus subtilis. Despite possessing distinct DNA-binding domains, both proteins form similar tetrameric assemblies when bound to their respective DNA operators. Structural analysis elucidates the process by which the CggR-regulated gapA operon is derepressed through the action of two effectors: fructose-1,6-bisphosphate and newly confirmed dihydroxyacetone phosphate. Our findings provide the first comprehensive understanding of the DNA binding mechanism of the SorC-family proteins, shedding new light on their functional characteristics.
Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Microscopía por Crioelectrón , Modelos Moleculares , Proteínas Represoras , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Unión Proteica , Multimerización de Proteína , ADN/química , ADN/metabolismo , Sitios de Unión , Regulación Bacteriana de la Expresión Génica , ADN Bacteriano/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Operón/genética , FructosadifosfatosRESUMEN
Purine nucleoside phosphorylase (PNP) is a well-known molecular target with potential therapeutic applications in the treatment of T-cell malignancies and/or bacterial/parasitic infections. Here, we report the design, development of synthetic methodology, and biological evaluation of a series of 30 novel PNP inhibitors based on acyclic nucleoside phosphonates bearing a 9-deazahypoxanthine nucleobase. The strongest inhibitors exhibited IC50 values as low as 19 nM (human PNP) and 4 nM (Mycobacterium tuberculosis (Mt) PNP) and highly selective cytotoxicity toward various T-lymphoblastic cell lines with CC50 values as low as 9 nM. No cytotoxic effect was observed on other cancer cell lines (HeLa S3, HL60, HepG2) or primary PBMCs for up to 10 µM. We report the first example of the PNP inhibitor exhibiting over 60-fold selectivity for the pathogenic enzyme (MtPNP) over hPNP. The results are supported by a crystallographic study of eight enzyme-inhibitor complexes and by ADMET profiling in vitro and in vivo.
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Inhibidores Enzimáticos , Purina-Nucleósido Fosforilasa , Humanos , Purina-Nucleósido Fosforilasa/metabolismo , Inhibidores Enzimáticos/química , CristalografíaRESUMEN
Targeting cyclin-dependent kinase 7 (CDK7) provides an interesting therapeutic option in cancer therapy because this kinase participates in regulating the cell cycle and transcription. Here, we describe a new trisubstituted pyrazolo[4,3-d]pyrimidine derivative, LGR6768, that inhibits CDK7 in the nanomolar range and displays favourable selectivity across the CDK family. We determined the structure of fully active CDK2/cyclin A2 in complex with LGR6768 at 2.6 Å resolution using X-ray crystallography, revealing conserved interactions within the active site. Structural analysis and comparison with LGR6768 docked to CDK7 provides an explanation of the observed biochemical selectivity, which is linked to a conformational difference in the biphenyl moiety. In cellular experiments, LGR6768 affected regulation of the cell cycle and transcription by inhibiting the phosphorylation of cell cycle CDKs and the carboxy-terminal domain of RNA polymerase II, respectively. LGR6768 limited the proliferation of several leukaemia cell lines, triggered significant changes in protein and mRNA levels related to CDK7 inhibition and induced apoptosis in dose- and time-dependent experiments. Our work supports previous findings and provides further information for the development of selective CDK7 inhibitors.
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Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes/genética , Fosforilación , Ciclo Celular , Pirimidinas/farmacología , Pirimidinas/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Aldo-keto reductase 1C3 (AKR1C3) catalyzes the reduction of androstenedione to testosterone and reduces the effectiveness of chemotherapeutics. AKR1C3 is a target for treatment of breast and prostate cancer and AKR1C3 inhibition could be an effective adjuvant therapy in the context of leukemia and other cancers. In the present study, steroidal bile acid fused tetrazoles were screened for their ability to inhibit AKR1C3. Four C24 bile acids with C-ring fused tetrazoles were moderate to strong AKR1C3 inhibitors (37-88% inhibition), while B-ring fused tetrazoles had no effect on AKR1C3 activity. Based on a fluorescence assay in yeast cells, these four compounds displayed no affinity for estrogen receptor-α, or the androgen receptor, suggesting a lack of estrogenic or androgenic effects. A top inhibitor showed specificity for AKR1C3 over AKR1C2, and inhibited AKR1C3 with an IC50 of â¼7 µM. The structure of AKR1C3·NADP+ in complex with this C-ring fused bile acid tetrazole was determined by X-ray crystallography at 1.4 Å resolution, revealing that the C24 carboxylate is anchored to the catalytic oxyanion site (H117, Y55); meanwhile the tetrazole interacts with a tryptophan (W227) important for steroid recognition. Molecular docking predicts that all four top AKR1C3 inhibitors bind with nearly identical geometry, suggesting that C-ring bile acid fused tetrazoles represent a new class of AKR1C3 inhibitors.
RESUMEN
The development of highly active and selective enzyme inhibitors is one of the priorities of medicinal chemistry. Typically, various high-throughput screening methods are used to find lead compounds from a large pool of synthetic compounds, and these are further elaborated and structurally refined to achieve the desired properties. In an effort to streamline this complex and laborious process, new selection strategies based on different principles have recently emerged as an alternative. Herein, we compare three such selection strategies with the aim of identifying potent and selective inhibitors of human carbonic anhydrase II. All three approaches, in situ click chemistry, phage-display libraries and synthetic peptide libraries, led to the identification of more potent inhibitors when compared to the parent compounds. In addition, one of the inhibitor-peptide conjugates identified from the phage libraries showed greater than 100-fold selectivity for the enzyme isoform used for the compound selection. In an effort to rationalize the binding properties of the conjugates, we performed detailed crystallographic and NMR structural analysis, which revealed the structural basis of the compound affinity towards the enzyme and led to the identification of a novel exosite that could be utilized in the development of isoform specific inhibitors.
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Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.
Asunto(s)
Cistatinas , Fasciola hepatica , Animales , Secuencia de Aminoácidos , Cistatinas/genética , Cistatinas/química , Disulfuros , Fasciola hepatica/genética , Filogenia , Proteínas del Helminto/química , Proteínas del Helminto/genéticaRESUMEN
Among non-covalent interactions, B-Hâ¯π and C-Hâ¯π hydrogen bonding is rather weak and less studied. Nevertheless, since both can affect the energetics of protein-ligand binding, their understanding is an important prerequisite for reliable predictions of affinities. Through a combination of high-resolution X-ray crystallography and quantum-chemical calculations on carbonic anhydrase II/carborane-based inhibitor systems, this paper provides the first example of B-Hâ¯π hydrogen bonding in a protein-ligand complex. It shows that the B-Hâ¯π interaction is stabilized by dispersion, followed by electrostatics. Furthermore, it demonstrates that the similar C-Hâ¯π interaction is twice as strong, with a slightly smaller contribution of dispersion and a slightly higher contribution of electrostatics. Such a detailed insight will facilitate the rational design of future protein ligands, controlling these types of non-covalent interactions.
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Anhidrasa Carbónica II , Sulfonamidas , Ligandos , Sulfanilamida , Cristalografía por Rayos XRESUMEN
Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.
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Proteínas de la Membrana , Nucleótidos Cíclicos , Ratones , Animales , Humanos , Nucleótidos Cíclicos/química , Ligandos , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas , Citocinas , InterferonesRESUMEN
3,5,7-Trisubstituted pyrazolo[4,3-d]pyrimidines have been identified as potent inhibitors of cyclin-dependent kinases (CDKs), which are established drug targets. Herein, we describe their further structural modifications leading to novel nanomolar inhibitors with strong antiproliferative activity. We determined the crystal structure of fully active CDK2/A2 with 5-(2-amino-1-ethyl)thio-3-cyclobutyl-7-[4-(pyrazol-1-yl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidine (24) at 1.7 Å resolution, confirming the competitive mode of inhibition. Biochemical and cellular assays in lymphoma cell lines confirmed the expected mechanism of action through dephosphorylation of retinoblastoma protein and RNA polymerase II, leading to induction of apoptosis. Importantly, we also revealed an interesting ability of compound 24 to induce proteasome-dependent degradation of cyclin K both in vitro and in a patient-derived xenograft in vivo. We propose that 24 has a dual mechanism of action, acting as a kinase inhibitor and as a molecular glue inducing an interaction between CDK12 and DDB1 that leads to polyubiquitination of cyclin K and its subsequent degradation.
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Antineoplásicos , Quinasas Ciclina-Dependientes , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina , Ciclinas/metabolismo , Humanos , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
The SorC/DeoR family is a large family of bacterial transcription regulators that are involved in the control of carbohydrate metabolism and quorum sensing. To understand the structural basis of DNA recognition, structural studies of two functionally characterized SorC/DeoR family members from Bacillus subtilis were performed: the deoxyribonucleoside regulator bsDeoR and the central glycolytic genes regulator bsCggR. Each selected protein represents one of the subgroups that are recognized within the family. Crystal structures were determined of the N-terminal DNA-binding domains of bsDeoR and bsCggR in complex with DNA duplexes representing the minimal operator sequence at resolutions of 2.3 and 2.1â Å, respectively. While bsDeoRDBD contains a homeodomain-like HTH-type domain, bsCggRDBD contains a winged helix-turn-helix-type motif. Both proteins form C2-symmetric dimers that recognize two consecutive major grooves, and the protein-DNA interactions have been analyzed in detail. The crystal structures were used to model the interactions of the proteins with the full DNA operators, and a common mode of DNA recognition is proposed that is most likely to be shared by other members of the SorC/DeoR family.
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Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Bacillus subtilis/química , Proteínas Bacterianas/química , Sitios de Unión , Cristalografía por Rayos X , ADN/metabolismo , Proteínas de Unión al ADN/química , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The 3H-pyrazolo[4,3-f]quinoline moiety has been recently shown to be a privileged kinase inhibitor core with potent activities against acute myeloid leukemia (AML) cell lines in vitro. Herein, various 3H-pyrazolo[4,3-f]quinoline-containing compounds were rapidly assembled via the Doebner-Povarov multicomponent reaction from the readily available 5-aminoindazole, ketones, and heteroaromatic aldehydes in good yields. The most active compounds potently inhibit the recombinant FLT3 kinase and its mutant forms with nanomolar IC50 values. Docking studies with the FLT3 kinase showed a type I binding mode, where the 3H-pyrazolo group interacts with Cys694 in the hinge region. The compounds blocked the proliferation of AML cell lines harboring oncogenic FLT3-ITD mutations with remarkable IC50 values, which were comparable to the approved FLT3 inhibitor quizartinib. The compounds also inhibited the growth of leukemia in a mouse-disseminated AML model, and hence, the novel 3H-pyrazolo[4,3-f]quinoline-containing kinase inhibitors are potential lead compounds to develop into anticancer agents, especially for kinase-driven cancers.
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Antineoplásicos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Células Tumorales Cultivadas , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.
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Proteínas Sanguíneas/metabolismo , Cistatinas/metabolismo , Sistema Digestivo/metabolismo , Ixodes/metabolismo , Garrapatas/metabolismo , Secuencia de Aminoácidos , Animales , Catepsina L/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Filogenia , ProteolisisRESUMEN
Cryptococcosis is an invasive infection that accounts for 15% of AIDS-related fatalities. Still, treating cryptococcosis remains a significant challenge due to the poor availability of effective antifungal therapies and emergence of drug resistance. Interestingly, protease inhibitor components of antiretroviral therapy regimens have shown some clinical benefits in these opportunistic infections. We investigated Major aspartyl peptidase 1 (May1), a secreted Cryptococcus neoformans protease, as a possible target for the development of drugs that act against both fungal and retroviral aspartyl proteases. Here, we describe the biochemical characterization of May1, present its high-resolution X-ray structure, and provide its substrate specificity analysis. Through combinatorial screening of 11,520 compounds, we identified a potent inhibitor of May1 and HIV protease. This dual-specificity inhibitor exhibits antifungal activity in yeast culture, low cytotoxicity, and low off-target activity against host proteases and could thus serve as a lead compound for further development of May1 and HIV protease inhibitors.
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Antifúngicos/química , Proteasas de Ácido Aspártico/antagonistas & inhibidores , Cryptococcus neoformans/enzimología , Proteínas Fúngicas/antagonistas & inhibidores , Antifúngicos/metabolismo , Antifúngicos/farmacología , Proteasas de Ácido Aspártico/genética , Proteasas de Ácido Aspártico/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hongos/efectos de los fármacos , VIH/enzimología , Proteasa del VIH/química , Proteasa del VIH/metabolismo , Simulación de Dinámica Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
This review describes recent progress in the design and development of inhibitors of human carbonic anhydrase IX (CA IX) based on space-filling carborane and cobalt bis(dicarbollide) clusters. CA IX enzyme is known to play a crucial role in cancer cell proliferation and metastases. The new class of potent and selective CA IX inhibitors combines the structural motif of a bulky inorganic cluster with an alkylsulfamido or alkylsulfonamido anchor group for Zn2+ ion in the enzyme active site. Detailed structure-activity relationship (SAR) studies of a large series containing 50 compounds uncovered structural features of the cluster-containing inhibitors that are important for efficient and selective inhibition of CA IX activity. Preclinical evaluation of selected compounds revealed low toxicity, favorable pharmacokinetics and ability to reduce tumor growth. Cluster-containing inhibitors of CA IX can thus be considered as promising candidates for drug development and/or for combination therapy in boron neutron capture therapy (BNCT).
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Compuestos de Boro/química , Anhidrasa Carbónica IX/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/química , Sitios de Unión , Compuestos de Boro/metabolismo , Compuestos de Boro/uso terapéutico , Anhidrasa Carbónica IX/metabolismo , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/metabolismo , Inhibidores de Anhidrasa Carbónica/uso terapéutico , Humanos , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Compuestos Organometálicos/química , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
Pharmacological inhibition of cyclin-dependent kinases has emerged as a possible treatment option for various cancer types. We recently identified substituted imidazo[1,2-c]pyrimidin-5(6H)-ones as inhibitors of cyclin-dependent kinase 2 (CDK2). Here, we report the synthesis of derivatives modified at positions 2, 3, 6 or 8 prepared using Suzuki-Miyaura cross-coupling, halogenation, Dimroth-type rearrangement and alkylation as the main synthetic methods. The compounds displayed micro- to submicromolar inhibition of CDK2/cyclin E activity. Binding of the most potent compound 3b to CDK2 was determined using isothermal titration calorimetry. The co-crystal structure of 3b in complex with fully active CDK2 was solved, revealing the binding mode of 3b in the ATP pocket and a hydrogen bonding interaction with hinge region residue Leu83. Evaluation against leukaemia cell lines revealed low cytotoxicity, which is in line with the high selectivity towards CDK2. This study demonstrates that substituted imidazo[1,2-c]pyrimidines can be exploited for future kinase inhibitor development.
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Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Imidazoles/química , Inhibidores de Proteínas Quinasas/síntesis química , Pirimidinas/química , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Sitios de Unión , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/metabolismo , Humanos , Enlace de Hidrógeno , Imidazoles/metabolismo , Imidazoles/farmacología , Simulación de Dinámica Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/metabolismo , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
While DNA encodes protein structure, glycans provide a complementary layer of information to protein function. As a prime example of the significance of glycans, the ability of the cell surface receptor CD44 to bind its ligand, hyaluronan, is modulated by N-glycosylation. However, the details of this modulation remain unclear. Based on atomistic simulations and NMR, we provide evidence that CD44 has multiple distinct binding sites for hyaluronan, and that N-glycosylation modulates their respective roles. We find that non-glycosylated CD44 favors the canonical sub-micromolar binding site, while glycosylated CD44 binds hyaluronan with an entirely different micromolar binding site. Our findings show (for the first time) how glycosylation can alter receptor affinity by shielding specific regions of the host protein, thereby promoting weaker binding modes. The mechanism revealed in this work emphasizes the importance of glycosylation in protein function and poses a challenge for protein structure determination where glycosylation is usually neglected.
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Receptores de Hialuranos/genética , Ácido Hialurónico/genética , Polisacáridos/genética , Conformación Proteica , Sitios de Unión/genética , Adhesión Celular/genética , Glicosilación , Humanos , Receptores de Hialuranos/ultraestructura , Espectroscopía de Resonancia Magnética , Unión Proteica/genética , Receptores de Superficie Celular/genéticaRESUMEN
Carbonic anhydrase IX (CAIX) is an enzyme expressed on the surface of cells in hypoxic tumors. It plays a role in regulation of tumor pH and promotes thus tumor cell survival and occurrence of metastases. Here, derivatives of the cobalt bis(dicarbollide)(1-) anion are reported that are based on substitution at the carbon sites of the polyhedra by two alkylsulfonamide groups differing in the length of the aliphatic connector (from C1 to C4, n=1-4), which were prepared by cobalt insertion into the 7-sulfonamidoalkyl-7,8-dicarba-nido-undecaborate ions. Pure meso- and rac-diastereoisomeric forms were isolated. The series is complemented with monosubstituted species (n=2). Synthesis by a direct method furnished similar derivatives (n=2, 3), which are chlorinated at the B(8,8') boron sites. All compounds inhibited CAIX with subnanomolar inhibition constants and showed high selectivity for CAIX. The best inhibitory properties were observed for the compound with n= 3 and two substituents present in rac-arrangement with Ki =20â pM and a selectivity index of 668. X-ray crystallography was used to study interactions of these compounds with the active site of CAIX on the structural level.
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Anhidrasa Carbónica IX/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/química , Cobalto/química , Complejos de Coordinación/química , Sulfonamidas/química , Sitios de Unión , Anhidrasa Carbónica IX/metabolismo , Inhibidores de Anhidrasa Carbónica/metabolismo , Inhibidores de Anhidrasa Carbónica/uso terapéutico , Dominio Catalítico , Complejos de Coordinación/metabolismo , Complejos de Coordinación/uso terapéutico , Cristalografía por Rayos X , Humanos , Conformación Molecular , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/patologíaRESUMEN
Azapeptide nitriles are postulated to reversibly covalently react with the active-site cysteine residue of cysteine proteases and form isothiosemicarbazide adducts. We investigated the interaction of azadipeptide nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a pathogen that causes the global neglected disease schistosomiasis. Azadipeptide nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We determined the crystal structure of SmCB1 in complex with an azadipeptide nitrile and analyzed the reaction mechanism using quantum chemical calculations. The data demonstrate that azadipeptide nitriles, in contrast to their carba counterparts, undergo a change from E- to Z-configuration upon binding, which gives rise to a highly favorable energy profile of noncovalent and covalent complex formation. Finally, azadipeptide nitriles were considerably more lethal than their carba analogs against the schistosome pathogen in culture, supporting the further development of this chemotype as a treatment for schistosomiasis.