RESUMEN
Fragment-based drug discovery (FBDD) has become a mainstream technology for the identification of chemical hit matter in drug discovery programs. To date, the food and drug administration has approved four drugs, and over forty compounds are in clinical studies that can trace their origins to a fragment-based screen. The challenges associated with implementing an FBDD approach are many and diverse, ranging from the library design to developing methods for identifying weak affinity compounds. In this article, we give an overview of current progress in fragment library design, fragment to lead optimisation and on the advancement in techniques used for screening. Finally, we will comment on the future opportunities and challenges in this field.
Asunto(s)
Diseño de Fármacos , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/tendencias , Bibliotecas de Moléculas Pequeñas/química , Cristalografía por Rayos X , Aprobación de Drogas , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/tendencias , Humanos , Espectroscopía de Resonancia Magnética , Unión ProteicaRESUMEN
ß-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.
Asunto(s)
Sitio Alostérico , Descubrimiento de Drogas , Glucosilceramidasa , Glucosilceramidasa/metabolismo , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/química , Humanos , Cristalografía por Rayos X , Relación Estructura-Actividad , Modelos Moleculares , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
Fragment-based drug discovery (FBDD) has become an established method for the identification of efficient starting points for drug discovery programs. In recent years, electrophilic fragment screening has garnered increased attention from both academia and industry to identify novel covalent hits for tool compound or drug development against challenging drug targets. Herein, we describe the design and characterization of an acrylamide-focused electrophilic fragment library and screening campaign against extracellular signal-regulated kinase 2 (ERK2) using high-throughput protein crystallography as the primary hit-finding technology. Several fragments were found to have covalently modified the adenosine triphosphate (ATP) binding pocket Cys166 residue. From these hits, 22, a covalent ATP-competitive inhibitor with improved potency (ERK2 IC50 = 7.8 µM), was developed.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos , Inhibidores de Proteínas Quinasas , Acrilamidas/química , Adenosina Trifosfato/química , Cristalografía por Rayos X , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Rayos XRESUMEN
ADAM (a disintegrin and metalloproteinase) 10 is a key member of the ADAM family of disintegrin and metalloproteinases which process membrane-associated proteins to soluble forms in a process known as 'shedding'. Among the major targets of ADAM10 are Notch, EphrinA2 and CD44. In many cell-based studies of shedding, the activity of ADAM10 appears to overlap with that of ADAM17, which has a similar active-site topology relative to the other proteolytically active ADAMs. The tissue inhibitors of metalloproteinases, TIMPs, have proved useful in the study of ADAM function, since TIMP-1 inhibits ADAM10, but not ADAM17; however, both enzymes are inhibited by TIMP-3. In the present study, we show that, in comparison with ADAM17 and the MMPs (matrix metalloproteinases), the N-terminal domains of TIMPs alone are insufficient for the inhibition of ADAM10. This knowledge could form the basis for the design of directed inhibitors against different metalloproteinases.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Inhibidores de Proteasas/farmacología , Inhibidor Tisular de Metaloproteinasa-1/aislamiento & purificación , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/aislamiento & purificación , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide/genética , Línea Celular Tumoral , Activación Enzimática , Humanos , Receptores de Hialuranos/metabolismo , Proteínas de la Membrana/genética , Mutación/genética , Inhibidores de Proteasas/aislamiento & purificación , Inhibidores de Proteasas/metabolismo , ARN Interferente Pequeño/genética , Solubilidad , Inhibidor Tisular de Metaloproteinasa-1/antagonistas & inhibidores , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-3/antagonistas & inhibidores , Inhibidor Tisular de Metaloproteinasa-3/genéticaRESUMEN
Metastatic cancer cells express Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) to degrade the extracellular matrix in order to facilitate migration and proliferation. Tissue Inhibitor of Metalloproteinase (TIMP)-2 is the endogenous inhibitor of the MMP. Here, we describe a novel and highly effective fusion strategy to enhance the delivery of TIMP-2 to MT1-MMP. We can reveal that TIMP-2 fused to the haemopexin +/- transmembrane domains of MT1-MMP (two chimeras named T2PEX+TM and T2PEX) are able to interact with MT1-MMP on the cell surface as well as intracellularly. In the case of T2PEX+TM, there is even a clear sign of MT1-MMP:T2PEX+TM aggregation by the side of the nucleus to form aggresomes. In vitro, T2PEX+TM and T2PEX suppress the gelatinolytic and invasive abilities of cervical carcinoma (HeLa) and HT1080 fibrosarcoma cancer cells significantly better than wild type TIMP-2. In mouse xenograft, we further demonstrate that T2PEX diminishes cervical carcinoma growth by 85% relative to the control. Collectively, our findings indicate the effectiveness of the fusion strategy as a potential targeted approach in cancer inhibition.
Asunto(s)
Fibrosarcoma/tratamiento farmacológico , Hemopexina/química , Metaloproteinasa 14 de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Inhibidor Tisular de Metaloproteinasa-2/farmacología , Secuencia de Aminoácidos , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Matriz Extracelular/metabolismo , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Células HeLa , Humanos , Metaloproteinasa 14 de la Matriz/metabolismo , Células Tumorales CultivadasRESUMEN
Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). TIMP molecules are made up of two domains: an N-terminal domain that associates with the catalytic cleft of the metalloproteinases (MP) and a smaller C-terminal domain whose role in MP association is still poorly understood. This work is aimed at investigating the role of the C-terminal domain in MP selectivity. In this study, we replaced the C-terminal domain of TIMP-1 with those of TIMP-2, -3 and -4 to create a series of "T1:TX" chimeras. The affinity of the chimeras against ADAM10, ADAM17, MMP14 and MMP19 was investigated. We can show that replacement of the C-terminal domain by those of other TIMPs dramatically increased the affinity of TIMP-1 for some MPs. Furthermore, the chimeras were able to suppress TNF-α and HB-EGF shedding in cell-based setting. Unlike TIMP-1, T1:TX chimeras had no growth-promoting activity. Instead, the chimeras were able to inhibit cell migration and development in several cancer cell lines. Our findings have broadened the prospect of TIMPs as cancer therapeutics. The approach could form the basis of a new strategy for future TIMP engineering.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Metaloproteinasas de la Matriz/química , Inhibidores Tisulares de Metaloproteinasas/farmacología , Secuencia de Aminoácidos , Western Blotting , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Similar a EGF de Unión a Heparina/química , Humanos , Metaloproteinasas de la Matriz/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The surface-anchored membrane type 1 matrix metalloproteinase (MT1-MMP) degrades a wide range of extracellular matrix components that includes collagens, laminins, fibronectin and the structural proteoglycan aggrecan. The enzyme modulates cell motility and plays an important role in tumour invasion and proliferation. We have previously designed a variant of tissue inhibitor of metalloproteinase (TIMP)-1 bearing a triple mutation (V4A+P6V+T98L, or N-TIMP-1(mt1)) that forms tight binary complex with the soluble catalytic domain of MT1-MMP [M.H. Lee, M. Rapti, G. Murphy, J. Biol. Chem. 278 (2003) 40224-40230]. Here, we report our latest findings on the cellular potency of this mutant against native MT1-MMP in cell-based environment. We show that N-TIMP-1(mt1) is a highly potent inhibitor against the ectodomain form of MT1-MMP (K(i) 9.53nM) with potential for further development as a therapeutic agent. The mutant is devoid of pro-MMP-2-activating capability but is highly effective in blocking MT1-MMP-mediated FITC-labelled collagen and gelatin film degradation in HTC75 fibrosarcoma and MCF7 breast cancer models. Most encouragingly, N-TIMP-1(mt1) is also effective against CD44 shedding in HTC75 cells and able to prevent tubule formation in human umbilical vascular endothelial cells (HUVEC) in a 3D fibrin gel model. We are interested in the development of the TIMPs as therapeutic agents against MT1-MMP related disorders such as cancers. Our findings here indicate the potential for the design of selective TIMPs with refined specificity and possibility for future therapeutic application.
Asunto(s)
Activación Enzimática/efectos de los fármacos , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/farmacología , Western Blotting , Línea Celular Tumoral , Activación Enzimática/fisiología , Ensayo de Inmunoadsorción Enzimática , Humanos , Receptores de Hialuranos/metabolismo , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Neovascularización Fisiológica/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/genéticaRESUMEN
Tissue inhibitor of metalloproteinases-2 (TIMP-2) is unique as it is the only member of the TIMP family that is involved in the cellular activation of promatrix metalloproteinase-2 (pro-MMP-2) by virtue of forming a trimolecular complex with membrane type 1 matrix metalloproteinase (MT1-MMP) on the cell surface. TIMP-4 is similar in structure to TIMP-2 but is unable to support the activation of the proenzyme. Several reports have highlighted the importance of the TIMP-2 C-terminal domain in the pro-MMP-2 activation complex; however, very little is known about the role of the extended AB loop of TIMP-2 in this mechanism even though it has been shown to interact with MT1-MMP. In this study we show by mutagenesis and kinetic analysis that it is possible to transfer the MT1-MMP binding affinity of the TIMP-2 AB loop to TIMP-4 but that its transplantation into TIMP-4 does not endow the inhibitor with pro-MMP-2 activating activity. However, transfer of both the AB loop and C-terminal domain of TIMP-2 to TIMP-4 generates a mutant that can activate pro-MMP-2 and so demonstrates that both these regions of TIMP-2 are important for the activation process.
Asunto(s)
Precursores Enzimáticos/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/química , Inhibidor Tisular de Metaloproteinasa-2/fisiología , Secuencia de Aminoácidos , Animales , Activación Enzimática , Precursores Enzimáticos/antagonistas & inhibidores , Humanos , Metaloproteinasa 14 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes Quiméricas/síntesis química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/fisiología , Estructura Terciaria de Proteína/genética , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Inhibidor Tisular de Metaloproteinasa-2/deficiencia , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidores Tisulares de Metaloproteinasas , Inhibidor Tisular de Metaloproteinasa-4RESUMEN
Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases, the ADAMs (a disintegrin and metalloproteinase) and the ADAM-TS (ADAM with thrombospondin repeats) proteinases. There are four mammalian TIMPs (TIMP-1 to -4), and each TIMP has its own profile of metalloproteinase inhibition. TIMP-4 is the latest member of the TIMPs to be cloned, and it has never been reported to be active against the tumor necrosis factor-alpha-converting enzyme (TACE, ADAM-17). Here we examined the inhibitory properties of the full-length and the N-terminal domain form of TIMP-4 (N-TIMP-4) with TACE and showed that N-TIMP-4 is a far superior inhibitor than its full-length counterpart. Although full-length TIMP-4 displayed negligible activity against TACE, N-TIMP-4 is a slow tight-binding inhibitor with low nanomolar binding affinity. Our findings suggested that the C-terminal subdomains of the TIMPs have a significant impact over their activities with the ADAMs. To elucidate further the molecular basis that underpins TIMP/TACE interactions, we sculpted N-TIMP-4 with the surface residues of TIMP-3, the only native TIMP inhibitor of the enzyme. Transplantation of only three residues, Pro-Phe-Gly, onto the AB-loop of N-TIMP-4 resulted in a 10-fold enhancement in binding affinity; the K(i) values of the resultant mutant were almost comparable with that of TIMP-3. Further mutation at the EF-loop supported our earlier findings on the preference of TACE for leucine at this locus. Drawing together our previous experience in TACE-targeted mutagenesis by using TIMP-1 and -2 scaffolds, we have finally resolved the mystery of the selective sensitivity of TACE to TIMP-3.
Asunto(s)
Metaloendopeptidasas/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Proteínas ADAM , Proteína ADAM17 , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Humanos , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidor Tisular de Metaloproteinasa-4RESUMEN
Membrane type 1-matrix metalloproteinase (MT1-MMP) is a zinc-dependent, membrane-associated endoproteinase of the metzincin family. The enzyme regulates extracellular matrix remodeling and is capable of cleaving a wide variety of transmembrane proteins. The enzymatic activity of MT1-MMP is regulated by endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMP). To date, four variants of mammalian TIMP have been identified. Whereas TIMP-2-4 are potent inhibitors against MT1-MMP, TIMP-1 displays negligible inhibitory activity against the enzyme. The rationale for such selectivity is hitherto unknown. Here we identify the surface epitopes that render TIMP-1 inactive against MT1-MMP. We show that TIMP-1 can be transformed into an active inhibitor against MT1-MMP by the mutation of a single residue, namely threonine 98 to leucine (T98L). The resultant mutant displayed inhibitory characteristics of a typical slow, tight binding inhibitor. The potency of the mutant could be further enhanced by the introduction of valine 4 to alanine (V4A) and proline 6 to valine (P6V) mutations. Indeed, the inhibitory profile of the triple mutant (V4A/P6V/T98L) is indistinguishable from those of other TIMPs. Our findings suggest that threonine 98 is critical in initiating MMP binding and complex stabilization. Our findings also provide a potential mechanistic explanation for MMP-TIMP selectivity.
Asunto(s)
Metaloendopeptidasas/antagonistas & inhibidores , Inhibidor Tisular de Metaloproteinasa-1/química , Secuencia de Aminoácidos , Epítopos/química , Epítopos/genética , Humanos , Técnicas In Vitro , Cinética , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Inhibidores de Proteasas/química , Inhibidores de Proteasas/inmunología , Inhibidores de Proteasas/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Homología de Secuencia de Aminoácido , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/inmunología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/química , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-3/química , Inhibidor Tisular de Metaloproteinasa-3/genética , Inhibidores Tisulares de Metaloproteinasas/química , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidor Tisular de Metaloproteinasa-4RESUMEN
Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous modulators of the zinc-dependent mammalian matrix metalloproteinases (MMPs) and their close associates, proteinases of the ADAM (a disintegrin and metalloproteinase) and ADAM with thrombospondin repeats families. There are four variants of TIMPs, and each has its defined set of metalloproteinase (MP) targets. TIMP-1, in particular, is inactive against several of the membrane-type MMPs (MT-MMPs), MMP-19, and the ADAM proteinase TACE (tumor necrosis factor-alpha-converting enzyme, ADAM-17). The molecular basis for such inactivity is unknown. Previously, we showed that TIMP-1 could be transformed into an active inhibitor against MT1-MMP by the replacement of threonine 98 residue with leucine (T98L). Here, we reveal that the T98L mutation has in fact transformed TIMP-1 into a versatile inhibitor against an array of MPs otherwise insensitive to wild-type TIMP-1; examples include TACE, MMP-19, and MT5-MMP. Using T98L as the scaffold, we created a TIMP-1 variant that is fully active against TACE. The binding affinity of the mutant (V4S/TIMP-3-AB-loop/V69L/T98L) (K (app)(i) 0.14 nm) surpassed that of TIMP-3 (K (app)(i) 0.22 nm), the only natural TIMP inhibitor of the enzyme. The requirement for leucine is absolute for the transformation in inhibitory pattern. On the other hand, the mutation has minimal impact on the MPs already well inhibited by wild-type TIMP-1, such as gelatinase-A and stromelysin-1. Not only have we unlocked the molecular basis for the inactivity of TIMP-1 against several of the MPs, but also our findings fundamentally modify the current beliefs on the molecular mechanism of TIMP-MP recognition and selectivity.
Asunto(s)
Treonina/química , Inhibidor Tisular de Metaloproteinasa-1/química , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Colagenasas/metabolismo , Cristalografía por Rayos X , Escherichia coli/metabolismo , Humanos , Cinética , Leucina/química , Metaloproteinasa 13 de la Matriz , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Secretadas , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Trombospondinas/química , Factores de TiempoRESUMEN
Tumor necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE, ADAM-17) is a zinc-dependent ADAM (a disintegrin and metalloproteinase) metalloproteinase (MP) of the metzincin superfamily. The enzyme regulates the shedding of a variety of cell surface-anchored molecules such as cytokines, growth factors, and receptors. The activities of the MPs are modulated by the endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMPs). Among the four mammalian TIMPs (TIMP-1 to -4), TACE is selectively inhibited by TIMP-3. The rationale for such selectivity is not fully understood. Here, we examine the molecular basis of TIMP-TACE selectivity using TIMP-2 as the scaffold. By systematically replacing the surface epitopes of TIMP-2 with those of TIMP-3 and a TIMP-1 variant V4S/TIMP-3 AB-loop/V69L/T98L, we created a novel TIMP-2 mutant that exhibits inhibitory potency almost equal to that of the TIMP-3. The affinity of the mutant with TACE is 1.49 nm, a marked improvement in comparison to that of the wild-type protein (Ki 893 nM). The inhibitory pattern of the mutant is typical of that of a slow, tight binding inhibitor. We identify phenylalanine 34, a residue unique to the TIMP-3 AB-loop, as a vital element in TACE association. Mutagenesis carried out on leucine 100 also upholds our previous findings that a leucine on the EF-loop is critical for TACE recognition. Replacement of the residue by other amino acids resulted in a dramatic decrease in binding affinity, although isoleucine (L100I) and methionine (L100M) are still capable of producing the slow, tight binding effect. Our findings here represent a significant advance toward designing tailor-made TIMPs for specific MP targeting.