Design of enzyme inhibitors using iterative protein crystallographic analysis

Appelt, K.; Bacquet, Russell J.; Bartlett, Charlotte A.; Booth, Carol L. J.; Freer, Stephan T.; Fuhry, Mary Ann M.; Gehring, Michael R.; Herrmann, Steven M.; Howland, Eleanor F.

doi:10.1021/jm00111a001

Cited by 226 publications

(81 citation statements)

References 3 publications

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“…There has been increasing interest in recent years in the possibility of using the wealth of structural information available about proteins in order to design suitable inhibitors which could act as specific targetted drugs or at least as potential lead compounds in the search for specific drugs; this field has been termed structure assisted (or rational) drug design [125]. Some notable successes have been achieved in this area, for example:-(a) the design of inhibitors of angiotensin-converting enzyme; these inhibitors are used extensively as anti-hypertensive agents, and (b) inhibitors of the HIV type 1 protease which has been shown to be essential for virion assembly and maturation and is therefore considered an excellent target for inhibitory drug design [126,127].…”

Section: Ligand Binding and Drug Designmentioning

confidence: 99%

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

Kelly

Price²

2000

CPPS

971

707

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Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as α-helix and β-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising "molten globule" intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications.

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Section: Ligand Binding and Drug Designmentioning

confidence: 99%

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

Kelly

Price²

2000

CPPS

971

707

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“…As a result, a number of (10)(11)(12)(13)(14)(15)(16). To further validate the in vivo significance of some of the highly significant markers that may be associated with chemoresponse to 5-FU-based therapy, a group of colorectal cancer patient specimens were selected.…”

Section: Introductionmentioning

confidence: 99%

Validation of biomarkers associated with 5-fluorouracil and thymidylate synthase in colorectal cancer

Formentini²,

Nakajima³

et al. 2008

Oncol Rep

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Abstract. Previous studies from our laboratory have identified a number of genes associated with chemosensitivity to 5-fluorouracil (5-FU) using an in vitro colon cancer cell line model. In this study, the in vivo significance of several marker genes in terms of prognostic potential was evaluated using colorectal cancer patient samples. Eight marker genes were selected based on their functional roles and significant fold changes in expression. They are SERTA domain containing 1 (SEI1), ribonucleotide reductase M2 polypeptide (RRM2), origin recognition complex, subunit 6 homolog-like (ORC6L), eukaryotic translation initiation factor 4E (EIF4E), thymidylate synthase (TS), SET and MYND domain containing 3 (SMYD3), Dickkopf homolog 4, and methyl-CpG binding domain protein 4 (MBD4). Forty-eight snap frozen clinical colorectal samples (24 normal and 24 paired colorectal cancer patient samples) were selected with detailed clinical followup information. cDNAs were synthesized and the expression levels of marker genes were quantified via qRT-PCR analysis. The statistical significance of these markers for disease prognosis was evaluated using the two-tailed paired Wilcoxon test. Survival curves were plotted according to the method of Kaplan-Meier and compared using the log-rank test. Based on the quantitative expression analysis, RRM2 (p=0.0001; 95% CI, 2.0-4.5), ORC6L (p=0.0001; 95% CI, 1.8-4.6), EIF4E (p=0.0002; 95% CI, 0.3-0.9), TS (p=0.0005; 95% CI, 0.7-2.2) and SMYD3 (p=0.0001; 95% CI, 0.8-1.5) were overexpressed in tumor tissues. However, the expression of SEI1 was decreased in tumors (p=0.02; 95% CI, 0.1-1.3), consistent with the function of SEI1 as a potential tumor suppressor. KaplanMeier survival analysis indicated that MBD4 is a significant prognostic factor for patient survival (p=0.03). MBD4 was a key protein involved in DNA methylation. The expression of TS was associated with tumor stage as it had a significantly higher expression level in UICC stage I and II compared to stage IV patients (p=0.03). MBD4 may be a potential novel prognostic marker for predicting patient survival for colorectal cancer.

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“…This is due in part to the lack of a complete structural description of IN, which is crucial for understanding its enzymatic activity. Such knowledge is the foundation of rational drug design (6).…”

mentioning

confidence: 99%

Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant^,

et al. 1999

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Six crystal structures of the core domain of integrase (IN) from avian sarcoma virus (ASV) and its active-site derivative containing an Asp64 f Asn substitution have been solved at atomic resolution ranging 1.02-1.42 Å. The high-quality data provide new structural information about the active site of the enzyme and clarify previous inconsistencies in the description of this fragment. The very high resolution of the data and excellent quality of the refined models explain the dynamic properties of IN and the multiple conformations of its disordered residues. They also allow an accurate description of the solvent structure and help to locate other molecules bound to the enzyme. A detailed analysis of the flexible active-site region, in particular the loop formed by residues 144-154, suggests conformational changes which may be associated with substrate binding and enzymatic activity. The pH-dependent conformational changes of the active-site loop correlates with the pH vs activity profile observed for ASV IN.Retroviruses, such as human immunodeficiency virus type 1 (HIV-1) 1 or avian sarcoma virus (ASV), encode in their genes three essential enzymes: reverse transcriptase (RT), protease (PR), and integrase (IN) (1). In rare cases a retrovirus such as feline immunodeficiency virus and equine infectious anemia virus also encodes dUTP-ase (2-4). The first three enzymes are considered to be primary targets for designing drugs against AIDS, because each enzyme is absolutely required for virus replication. Although the search for therapeutically suitable inhibitors has been successful for RT and PR and a number of drugs against these enzymes are already in use (5), no such drugs targeted against IN are yet available. This is due in part to the lack of a complete structural description of IN, which is crucial for understanding its enzymatic activity. Such knowledge is the foundation of rational drug design (6).A molecule of retroviral IN contains approximately 300 amino acids, and it comprises three domains: the zincbinding N-terminal domain, the catalytic core domain, and the DNA-binding C-terminal domain. IN catalyzes the incorporation of reverse-transcribed viral DNA into the host genome in two steps, processing and joining (1,7,8), both of which involve nucleophilic attack by a hydroxyl group on a DNA phosphate. In the processing step, a water molecule attacks near the end of the viral DNA, displacing two nucleotides from the 3′ ends of each of the viral DNA strands. In the joining step, each exposed viral DNA ribose 3′-OH is activated to attack the host DNA at a relatively nonspecific location with a separation of five or six nucleotides, thereby inserting viral DNA into the host genome. In vitro, these reactions require only IN, metal cations, and DNA, although other proteins play a role in vivo.The structures of the N-terminal domain (9, 10) and C-terminal domain (11, 12) of HIV IN have been determined by nuclear magnetic resonance (NMR) spectroscopy, whereas the structure of the catalytic core domain ha...

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Design of enzyme inhibitors using iterative protein crystallographic analysis

Cited by 226 publications

References 3 publications

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

Validation of biomarkers associated with 5-fluorouracil and thymidylate synthase in colorectal cancer

Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant^,

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Design of enzyme inhibitors using iterative protein crystallographic analysis

Cited by 226 publications

References 3 publications

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

The Use of Circular Dichroism in the Investigation of Protein Structure and Function

Validation of biomarkers associated with 5-fluorouracil and thymidylate synthase in colorectal cancer

Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant,

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Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant^,