A family of 4-heteroaryl-2-amino-pyrimidine CDK2 inhibitor lead compounds was discovered with the new database-mining program LIDAEUS through in silico screening. Four compounds with IC(50) values ranging from 17 to 0.9 microM were selected for X-ray crystal analysis. Two distinct binding modes are observed, one of which resembles the hydrogen bonding pattern of bound ATP. In the second binding mode, the ligands trigger a conformational change in the activation T loop by inducing movement of Lys(33) and Asp(145) side chains. The family of molecules discovered provides an excellent starting point for the design and synthesis of tight binding inhibitors, which may lead to a new class of antiproliferative drugs.

We report the cloning, overexpression, kinetic analysis, and modeling of the tertiary structure of an unusual plant cysteine proteinase. Ananain (EC 3.4.22.31), from Ananas comosus (pineapple) is distinguished from all other cysteine proteinases in the papain superfamily by having a unique combination of acidic amino acids. As well as lacking the acidic residue immediately preceding the active site histidine (position 158 in papain), it also lacks the extensive surface network of acidic residues that were postulated to compensate for the loss of charge at position 158 in mammalian cathepsins. Ananain has the fewest acidic residues, so far reported, of any plant cysteine proteinase, but two of the carboxyl residues (E50 and E35) postulated to have an enabling role in catalysis, the so-called "electrostatic switch", remain conserved. Comparisons of the kinetics of recombinant wild-type ananain with E50A and E35A mutants proves that these charged groups are not essential for catalysis. Hence this research does not confirm the presence of an electrostatic switch in this cysteine proteinase, and the role of acidic residues in the enhancement of catalytic competence in these enzymes is discussed in light of this new evidence.

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240BIOCHEMICAL SOCIETY TRANSACTIONS on the aggregation to a fine floc of a-lactalbumin in acid media under controlled conditions of ionic strength and temperature [28, 291. Both products of the process, purified alactalbumin and a-lactalbumin-depleted whey protein, have potential as nutritional or functional food ingredients. Scaleup of floc separation presents some difficulties that have yet to be resolved.The most widely used molecular separation technology in the dairy industry is membrane filtration. Within the past 20 years, use of this technology for production of whey protein concentrates or for concentration of milk has become widespread, and many technical improvements have been made which have greatly enhanced membrane performance [3O]. To date, however, application is confined predominantly to the separation in the permeate stream of small molecules such as water and lactose, and early aspirations to apply the technology to protein-protein separations have not been realised. A major characteristic of the operation of membranes with complex materials such as milk is the occurrence of 'concentration polarization', the build-up of rejected particles/molecules (mainly protein, fat) at the surface of the membrane, leading to reduced membrane flux and gradual blockage of the membrane pores (fouling). This alters permeation characteristics in a way that can reduce greatly the molecular exclusion limits of the membrane and thereby impair resolution of protein mixtures. It has been suggested that a solution to this problem may be in sight with the advent of a new generation of membranes of ceramic composition (ZrO,, AI,O,, CsI) [3 1,321. A feature of these membrane systems is pore homogeneity and high tangential flow velocities. This impairs concentration polarization and maintains pore fidelity during continuous operation. Currently, these membranes are manufactured with pore sizes of 0.08-5 pm, for operation in the microfiltration range, and have been used for clarification of fine suspensions or separation of bacteria and large proteins such as immunoglobulins [ 3 1-34]. More widespread application to protein-protein separations can be expected, in particular where a substantial differential in protein size exists. Separations which exploit the aggregation behaviour of caseins can be anticipated, leading to a new approach to whey protein-casein separation or to production of individual caseins such as P-casein [ 3 11.In conclusion, technology for new protein-protein separations in the dairy industry is in its infancy, but recent advances in technology and the potential for enhancing the value of milk proteins through separation, should lead to the emergence of new milk protein products in the not-toodistant future.