By site-directed mutagenesis, TEM-1 beta-lactamase was altered to contain single amino acid changes of E104K, R164S, and E240K, in addition to double changes of E104K/R164S or R164S/E240K and the triple change of E104K/R164S/E240K. Hydrolysis rates for cephaloridine and benzylpenicillin were lowered at least 1 order of magnitude for all enzymes containing R164S substitutions. All mutant enzymes exhibited increased kcat values for beta-lactam antibiotics containing an aminothiazole oxime side chain. Hydrolysis of ceftazidime was most affected, with kcat values increased 3-4 orders of magnitude in all enzymes with the substituted R164S moiety. Km values decreased for all substrates except ceftazidime in the enzymes with multiple mutations. Aztreonam was most affected, with Km values lowered 23-56-fold in the enzymes bearing multiple mutations. When the crystal structures of aztreonam and related monobactams were studied and projected into an active-site model of the PC1 beta-lactamase, it became apparent that the two lysine residues might serve equivalent roles by interacting with the carboxylate of the aminothiazole oxime side chain. Hydrogen-bonding interactions involving the oxime and N7 of the lysine, particularly Lys-104, may also be important in some antibiotics. Ser-164 apparently serves an indirect role, since it is somewhat distant from the active-site cleft.

The crystallographic structures of the ternary complexes of human a-thrombin with hirugen (a sulfated hirudin fragment) and the small-molecule active site thrombin inhibitors BMS-186282 and BMS-189090 have been determined at 2.6 and 2.8 A. In both cases, the inhibitors, which adopt verysimilar bound conformations, bind in an antiparallel P-strand arrangement relative to the thrombin main chain in a manner like that reported for PPACK, D-Phe-Pro-Arg-CH,Cl. They do, however, exhibit differences in the binding of the alkyl guanidine moiety in the specificity pocket. Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the binding pocket. Although PPACK forms covalent bonds to both serine and the histidine of the catalytic triad of thrombin, neither BMS-186282 nor BMS-189090 bind covalently and only BMS-186282 forms a hydrogen bond to the serine of the catalytic triad. Both inhibitors bind with high affinity (K, = 79 nM and 3.6 nM, respectively) and are highly selective for thrombin over trypsin and other serine proteases.Keywords: crystallography; human a-thrombin-inhibitor complexes; structure-based drug designThe serine protease thrombin occupies a central position in the blood coagulation cascade. It cleaves circulating fibrinogen to fibrin monomers, which then polymerize and are covalently linked by thrombin-activating factor XIIIa, which is also a prodReprint requests to: Mary E Malley, Department of Solid State Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, New Jersey 08543-4000; e-mail: malley@bms.com.Present address: Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, Indiana 47907. uct of thrombin activation. In addition, thrombin activates several other coagulation and plasma factors, such as factors V, VIII, protein C, and protein S. Thrombin also induces platelet aggregation and promotes the proliferation of endothelial cells, the liberation of tissue plasminogen activator, and the contraction and dilation of blood vessels. Selective inhibition of thrombin may result in efficient control of various pathophysiologic states, such as thrombosis and arteriosclerosis, and aid in the prevention of myocardial infarction (Berliner 1992; Fenton, 1986; Fenton et al., 1991). Abbreviations: BMS-186282, [S-(R*,R*)]-4-[Aminoiminomethyl)-amino]-N-[[l-[3-hydy-2-[(2-naphthalenyls~fony~)~no]-l-oxopropyl]- 2-pyrrolidinyl]methyI]butanamide; BMS-189090, [S-(R*,R*)]-l-(Aminoiminomethyl)-N-[[l-[N-[(2-naphthalenylsulfonyl)-~-seryl]-2-pyrrolidinyl]-Human a-thrombin consists of two polypeptide chains, A and B, connected through a single disulfide bond. The A chain has 36 residues (after the loss of a tridecapeptide during activation with prothrombin) and the B chain has 259 residues. The B chain is glycosylated at Asn 60G and contains the active site residues His 57, Asn 102, and Ser 195. The numbering scheme for the thrombin residues in this paper are assigned by homology with chymotrypsin as d...

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Investigated the association between various depression assessment methods in 38 adults with mild or moderate mental retardation, half of whom had relatively high and the other half had relatively low depression screening scores. Measures included a standard psychiatric interview (Diagnostic Interview for Children and Adolescents), an informant rating scale (Reiss Screen for Maladaptive Behavior), and a self-report measure (Self-Report Depression Questionnaire). Association between measures was generally low, yielding discordant classification results. Potential reasons for these discrepancies were offered, and implications for clinical and research assessment of mood disorders in mental retardation were discussed.

An isolate of Serratia marcescens that produced both an inducible chromosomal and a plasmid-mediated TEM-1 _j-lactamase was resistant to ampicillin and amoxicillin and also demonstrated decreased susceptibility to extended-spectrum P-lactam antibiotics (ESBAs). Clavulanic acid did not lower the MICs of the ESBAs, but it decreased the MICs of the penicillins. The TEM-l-producing plasmid was transferred to a more susceptible S. marcescens strain that produced a well-characterized inducible chromosomal 13-lactamase. The MICs of the ESBAs remained at a low level for the transconjugant. Ampicillin and amoxicillin, which were good substrates for the plasmid-mediated enzyme, were not well hydrolyzed by the chromosomal enzymes; the ESBAs were hydrolyzed slowly by all the enzymes. When each of the S. marcescens strains was grown with these 3-lactam antibiotics, at least modest increases in chromosomal 3-lactamase activity were observed. When organisms were grown in the presence of clavulanic acid and an ESBA, no enhanced induction was observed. The increases in the MICs of the ESBAs observed for the initial clinical isolate may have been due to a combination of low inducibility, slow hydrolysis, and differences in permeability between the S. marcescens isolates. When clavulanic acid and a penicillin were added to strains that produced both a plasmid-mediated TEM and a chromosomal P-lactamase, much higher levels of chromosomal j3-lactamase activity were present than were observed in cultures induced by the penicillin alone. This was due to the higher levels of penicillin that were available for induction as a result of inhibition of the TEM enzyme by clavulanate.Resistance to P-lactam antibiotics is caused to a great extent by the presence of P-lactamases, enzymes that are capable of destroying the antibacterial activities of many of these agents. Clavulanic acid has become a useful supplement to penicillins that are susceptible to hydrolysis by plasmid-mediated P-lactamases. This 3-lactamase inactivator works well when it is combined with amoxicillin or ticarcillin to treat infections caused by bacteria that produce many kinds of penicillinases or P-lactamases with broadspectrum hydrolyzing activities (1, 17). However, recent reports indicate that occasional antagonism of penicillin activity has been observed when antibiotic combinations with clavulanic acid are added to selected gram-negative rods (19,21). In certain isolates, the lack of synergy observed has been attributed to the induction of chromosomal cephalosporinases (10, 12, 22), P-lactamases that are not well inhibited by clavulanate. Although most studies have included organisms that produce only a single inducible chromosomal cephalosporinase, some resistant isolates have been identified as strains that produce multiple P-lactamases (8,19 Bio-Rad (Richmond, Calif.).Organisms. S. marcescens SC 15071, which produces cephalosporinase S1 and the plasmid-mediated TEM-1 P-lactamase (plasmid pSVA071), was from a group of clinical isolates selected at the Seattle Ve...