Masaichi Warizaya

Inhibitor-induced structural change of the active site of human poly(ADP-ribose) polymerase

The crystal structure of human recombinant poly(ADP‐ribose) polymerase (PARP) complexed with a potent inhibitor, FR257517, was solved at 3.0 Å resolution. The fluorophenyl part of the inhibitor induces an amazing conformational change in the active site of PARP by motion of the side chain of the amino acid, Arg878, which forms the bottom of the active site. Consequently, a corn‐shaped hydrophobic subsite, which consists of the side chains of Leu769, Ile879, Pro881, and the methylene chain of Arg878, newly emerges from the well‐known active site.

Discovery of quinazolinone and quinoxaline derivatives as potent and selective poly(ADP-ribose) polymerase-1/2 inhibitors.

Two classes of quinazolinone derivatives and quinoxaline derivatives were identified as potent and selective poly(ADP‐ribose) polymerase‐1 and 2 (PARP‐1) and (PARP‐2) inhibitors, respectively. In PARP enzyme assays using recombinant PARP‐1 and PARP‐2, quinazolinone derivatives displayed relatively high selectivity for PARP‐1 and quinoxaline derivatives showed superior selectivity for PARP‐2. SBDD analysis via a combination of X‐ray structural study and homology modeling suggested distinct interactions of inhibitors with PARP‐1 and PARP‐2. These findings provide a new structural framework for the design of selective inhibitors for PARP‐1 and PARP‐2.

Lead Generation and Examples: Opinion Regarding How to Follow Up Hits

In fragment-based drug discovery (FBDD), not only identifying the starting fragment hit to be developed but also generating a drug lead from that starting fragment hit is important. Converting fragment hits to leads is generally similar to a high-throughput screening (HTS) hits-to-leads approach in that properties associated with activity for a target protein, such as selectivity against other targets and absorption, distribution, metabolism, excretion, and toxicity (ADME/Tox), and physicochemical properties should be taken into account. However, enhancing the potency of the fragment hit is a key requirement in FBDD, unlike HTS, because initial fragment hits are generally weak. This enhancement is presently achieved by adding additional chemical groups which bind to additional parts of the target protein or by joining or combining two or more hit fragments; however, strategies for effecting greater improvements in effective activity are needed. X-ray analysis is a key technology attractive for converting fragments to drug leads. This method makes it clear whether a fragment hit can act as an anchor and provides insight regarding introduction of functional groups to improve fragment activity. Data on follow-up chemical synthesis of fragment hits has allowed for the differentiation of four different strategies: fragment optimization, fragment linking, fragment self-assembly, and fragment evolution. Here, we discuss our opinion regarding how to follow up on fragment hits, with a focus on the importance of fragment hits as an anchor moiety to so-called hot spots in the target protein using crystallographic data.

Advances in fragment-based drug discovery platforms

Background: Fragment-based drug discovery (FBDD) has been established as a powerful alternative and complement to traditional high-throughput screening techniques for identifying drug leads. At present, this technique is widely used among academic groups as well as small biotech and large pharmaceutical companies. In recent years, > 10 new compounds developed with FBDD have entered clinical development, and more and more attention in the drug discovery field is being focused on this technique. Objective: Under the FBDD approach, a fragment library of relatively small compounds (molecular mass = 100 – 300 Da) is screened by various methods and the identified fragment hits which normally weakly bind to the target are used as starting points to generate more potent drug leads. Because FBDD is still a relatively new drug discovery technology, further developments and optimizations in screening platforms and fragment exploitation can be expected. This review summarizes recent advances in FBDD platforms and discusses the factors important for the successful application of this technique. Conclusion: Under the FBDD approach, both identifying the starting fragment hit to be developed and generating the drug lead from that starting fragment hit are important. Integration of various techniques, such as computational technology, X-ray crystallography, NMR, surface plasmon resonance, isothermal titration calorimetry, mass spectrometry and high-concentration screening, must be applied in a situation-appropriate manner.

4-Hydroxypyridazin-3(2H)-one Derivatives as Novel d-Amino Acid Oxidase Inhibitors

D-Amino acid oxidase (DAAO) catalyzes the oxidation of d-amino acids including d-serine, a coagonist of the N-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3(2H)-one derivatives as novel DAAO inhibitors with high potency and substantial cell permeability using fragment-based drug design. Comparisons of complex structures deposited in the Protein Data Bank as well as those determined with in-house fragment hits revealed that a hydrophobic subpocket was formed perpendicular to the flavin ring by flipping Tyr224 in a ligand-dependent manner. We investigated the ability of the initial fragment hit, 3-hydroxy-pyridine-2(1H)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2-phenylethyl)pyridine-2(1H)-one exhibited the predicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme- and cell-based assays. We further designed and synthesized 4-hydroxypyridazin-3(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one was found to be effective against MK-801-induced cognitive deficit in the Y-maze.

Structure of a high-resolution crystal form of human triosephosphate isomerase: improvement of crystals using the gel-tube method

Crystals of human triosephosphate isomerase with two crystal morphologies were obtained using the normal vapour-diffusion technique with identical crystallization conditions. One had a disordered plate shape and the crystals were hollow (crystal form 1). As a result, this form was very fragile, diffracted to 2.8 A resolution and had similar crystallographic parameters to those of the structure 1hti in the Protein Data Bank. The other had a fine needle shape (crystal form 2) and was formed more abundantly than crystal form 1, but was unsuitable for structure analysis. Since the normal vapour-diffusion method could not control the crystal morphology, gel-tube methods, both on earth and under microgravity, were applied for crystallization in order to control and improve the crystal quality. Whereas crystal form 1 was only slightly improved using this method, crystal form 2 was greatly improved and diffracted to 2.2 A resolution. Crystal form 2 contained a homodimer in the asymmetric unit, which was biologically essential. Its overall structure was similar to that of 1hti except for the flexible loop, which was located at the active centre Lys13.

Improving quality and harvest period of protein crystals for structure-based drug design: effects of a gel and a magnetic field on bovine adenosine deaminase crystals.

The overall crystal quality as well as the harvest period of bovine adenosine deaminase containing a zinc ion at the active centre has been compared in three different environments: crystallization as a control, crystallization with agarose gel and crystallization in a high magnetic field. In crystallization with agarose gel, the probability of obtaining high-quality crystals was somewhat increased, but the harvest period was elongated. On the other hand, in crystallization in a magnetic field, the probability of obtaining high-quality crystals was greatly increased. Furthermore, the harvest period for crystal growth in a magnetic field was much shorter than that with agarose gel.

Improving the quality of protein crystals using stirring crystallization

Recent reports state that a high magnetic field improves the crystal quality of bovine adenosine deaminase (ADA) with an inhibitor [Kinoshita et al.: Acta Cryst. D59 (2003) 1333]. In this paper, we examine the effect of stirring solution on ADA crystallization using a vapor-diffusion technique with rotary and figure-eight motion shakers. The probability of obtaining high-quality crystals is increased with stirring in a figure-eight pattern. Furthermore, rotary stirring greatly increased the probability of obtaining high-quality crystals, however, nucleation time was also increased. The crystal structure with the inhibitor was determined at a high resolution using a crystal obtained from a stirred solution. These results indicate that stirring with simple equipment is as useful as the high magnetic field technique for protein crystallization.

Solution stirring initiates nucleation and improves the quality of adenosine deaminase crystals.

Crystals of bovine adenosine deaminase (ADA) grown over a two-week period in the presence of an inhibitor (ADA complex) were found to be of low quality for X-ray diffraction analysis. Furthermore, ADA incubated in the absence of an inhibitor (ADA native) did not form any crystals using conventional crystallization methods. A solution-stirring technique was used to obtain high-quality ADA complex and ADA native crystals. The crystals obtained using this technique were found to be of high quality and were shown to have high structural resolution for X-ray diffraction analyses. The results reported here indicate that the solution-stirring technique promotes nucleation and improves the quality of protein crystals.

Protein crystallization by combining laser irradiation and solution-stirring techniques

Bovine adenosine deaminase in the absence of an inhibitor (free-ADA) does not form crystals when using conventional crystallization methods. Using a solution-stirring technique, we recently succeeded in generating a small number of free-ADA crystals. In this paper, we demonstrate the combination of laser-irradiated growth and stirring (COLAS). This technique was found to be useful for controlling crystal nucleation and growth, which led to the production of a much larger number of high-quality free-ADA crystals.