random mutagenesis phage display to introduce mutations into the truncated binding domain. In this technique, genes are modified randomly and in serted into phages (bacterial viruses), Taking a key step in efforts to shrink which express the corresponding pro proteins to the size of small organic mol teins on their surfaces. Each protein ecules, scientists at Genentech, South that binds to a target with high affinity San Francisco, have removed almost half is isolated and identified by sequenc a protein's binding domain and then re ing the gene from its associated phage. Braisted and Wells modified the stored the domain's structural stability and binding capacity. The research may protein A domain in three steps—first have implications for drug design and revising an area that became exposed to solvent when the helix was re aid protein-folding studies. "A folded structural unit of a protein moved, next stabilizing the interface can be put on a serious diet and re between the two remaining helices, duced in size without sacrificing bind- and then optimizing residues in the an tibody-binding site. δ The final 33-residue, I two-helix structure ^ bound to antibody I with about one| fourth the affinity of ^ the natural protein I domain. S Genentech scien| tist Brian Cunning§" ham, Wells, and co§ workers used a simI ilar strategy last §. year to reduce the | hormone atrial na| triuretic peptide | from 28 residues to 1-15 while retaining high potency. Other groups have also devised miniprotein Binding domain of protein A (purple) in complex with antibodydomains in other fragment (blue). Braisted and Wells restored domain's structural ways. integnty and binding after deleting helix 3. "The paper by Braisted and Wells mg," says staff scientist James A. Wells represents another particularly excit of Genentech's department of protein ing demonstration that protein do engineering. He and postdoctoral fel mains need not be as large as has low Andrew C. Braisted minimized the been traditionally thought," says a protein domain using mutagenesis and commentary in PNAS by professor rational design techniques [Proc. Natl. William F. DeGrado and research as Acad. Sei. USA, 93, 5688 (1996)]. sistant professor Tobin R. Sosnick of Braisted and Wells removed a pure the department of biochemistry and ly structural α-helix from the 59-resi- biophysics at the University of Penn due antibody-binding domain of pro sylvania School of Medicine. tein A, a popular reagent for purifying The work may be applicable to antibodies. The domain's two other drug design. "Today, there is no good α-helices form its binding site. The de way to be able to take a protein phar letion made the protein domain fall maceutical and rationally reduce it to apart, destroying its distinct structure a small molecule," Wells says. "What and its ability to bind antibody. we've done is to take a protein and The researchers reinstated the do bring it to a realm where it's amena main's structural integrity and a sub ble to manipulation by chemical syn stantial degree of its binding affinity by thesis, which greatly expands the di using a combinatorial technique called versity that can be introduced." He
Minimized protein still binds antibody
hopes to reduce the size of protein binding domains further and to use the minimized domains as models to de sign small organic molecule mimics. Protein minimization may also aid studies of protein folding. "Folding has been difficult to study in part because the large size of proteins has hampered both computational and experimental work," write DeGrado and Sosnick. "This 33-residue structured protein should serve as an attractive starting point for the systematic study of both kinetics and thermodynamics of pro tein folding in a much more fine-tuned manner than has been possible with site-directed mutagenesis," a biological method of modifying proteins. Stu Borman
DuPont loses Benlate birth defect trial A Florida jury has awarded $4 million to the family of a six-year-old boy who was born without eyes—a condition the family claims was caused by prena tal exposure to DuPont's Benlate DF fungicide. The four-week trial was held in the 11th Circuit Court in Dade County. The lawsuit is the first to come to trial linking birth defects to Benlate. For more than three years, DuPont has been wag ing a legal battle against farmers claim ing the fungicide damaged or killed crops. The plaintiffs' lead attorney, James L. Ferraro, says the verdict could pave the way for lawsuits in 40 other cases of deformed or missing eyes that victims blame on Benlate. Stacey J. Mobley, DuPont senior vice president for external affairs, calls the verdict "a blow to science and to our jury system. Birth defects are a human tragedy. The jury in this case was none theless faced with the challenge of looking beyond that tragedy to deter mine whether DuPont's product, Ben late, bears any connection with John Castillo's birth defect. In this instance, the challenge was not met." DuPont plans to appeal the verdict. During her pregnancy, the boy's mother took frequent walks along the edge of Pine Island Farms in Miami, which used the fungicide. At one time, Ferraro says, she was sprayed by a "misty cloud" that covered more than 50% of her body. JUNE 17,1996 C&EN
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NEWS OF THE WEEK She did not connect her son's birth defect to this exposure "until a 1993 BBC [broadcast] report called it to her attention/' Ferraro explains. He says the BBC found a high number of children born with deformed or missing eyes in farming areas where Benlate was used. Ferraro adds that studies have found that pregnant rats exposed to benomyl, the active ingredient in Benlate, give birth to offspring with no eyes. And he says that studies conducted by the University of California, the Environmental Protection Agency, and DuPont suggest the link in humans. DuPont disputes the links that Ferraro claims exist. A spokesman says pesticides are tested at extremely high rates, and rats have a predisposition to eye defects. Moreover, "subsequent to those studies," Benlate was approved for sale by EPA. "There is no credible scientific evidence linking the use of Benlate to human health effects," says Mobley. "Time
and again, scientific authorities and governmental agencies worldwide have independently come to this conclusion. Until today, courts had arrived at the same conclusion, dismissing lawsuits in view of the total absence of scientific support for the arguments of plaintiffs' lawyers." Meanwhile, DuPont is continuing to defend Benlate DF against about 80 pending lawsuits, most of which allege crop damage. DuPont paid out $500 million to settle such claims before studies in November 1992 that it said exonerated the fungicide. Since then, DuPont has battled lawsuits with mixed results, winning and losing about the same number of cases. Benlate DF—the granular, dryflowable form of Benlate—was pulled from the market in 1991. DuPont continues to sell the wettable-powder form, Benlate WP. It will not disclose sales figures, but says Benlate WP sells well around the world. Elisabeth Kirschner
Monsanto and Chevron settle bias claims Just as their case was to go to trial, 43 former sales employees of a Chevron Chemical unit—who lost their jobs when Monsanto bought the unit— reached an $18.3 million settlement of claims of age, race, and disability discrimination against the two companies. Judge George F. Gunn Jr., of the U.S. District Court for the Eastern District of Missouri, which is in St. Louis, approved the agreement last week. The plaintiffs worked at Chevron's Ortho lawn and garden chemicals unit. Monsanto bought Ortho in 1993 and merged it into its Solaris consumer agricultural chemicals unit. Monsanto retained only 300 of some 400 Ortho employees, including 85 of Ortho's 152 sales employees. The remainder were let go or found other employment at Chevron. Payments to the 43 former sales employees will range from $201,000 to $722,000. The workers sued in 1994 for $30 million to $40 million in damages. All 43, who ranged in age from 41 to 58 at the time of dismissal, claimed age discrimination. The wide variation in payouts stems from compensation to four black employees, who also alleged race discrimination, and to one worker who was on 8
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a leave of absence in 1993 because of a brain tumor and who claimed disability discrimination. The award range also reflects calculations of actual lost wages and obtaining new jobs by some of the 43. Plaintiffs' attorney Stephen J. Snyder of Winthrop & Weinstine, Minneapolis, says the agreement upholds employees' rights. It "proves that employers must treat employees fairly regardless of their age, race, or disability." However, Monsanto and Chevron strongly deny any discrimination. Solaris President Janice V. Novak says, "No one at Monsanto or Solaris had done anything to discriminate against any person or group. We decided to settle these claims to avoid the expense and uncertainty of litigation." Adds Chevron Chemical Senior Vice President Darry Callahan: "Chevron does not discriminate and is committed to the fair and sensitive treatment of all employees." Monsanto will pay all the $18.3 million. "When we acquired the Ortho business from Chevron, we agreed to indemnify Chevron," explains a Monsanto spokeswoman. The $18.3 million not only compensates the plaintiffs, but also includes
their attorneys' fees and the costs of the Equal Employment Opportunity Commission (EEOC), which intervened on their behalf. And under the agreement, Solaris and Chevron Chemical's remaining agricultural chemicals unit must train supervisory personnel on compliance with antidiscrimination laws and must consult EEOC in preparing training materials. EEOC also will monitor employment practices at both units for the next three years. Marc Reisch
EPA sets rules for Risk Management Program The Environmental Protection Agency has issued final regulations for the Risk Management Program mandated by the 1990 Clean Air Act. Facilities will have three years to come into compliance with the rules. Aimed at preventing accidents involving hazardous chemicals and keeping local communities informed about what chemicals plants are using, the program has the same goals as and complements the Occupational Safety & Health Administration's (OSHA) Process Safety Management Standard. Under the Risk Management Program, EPA will require many facilities that handle hazardous chemicals to publish emergency release plans, worst-case accident scenarios, and fiveyear accident histories. The rules cover chemical manufacturers, drinking water systems, wastewater treatment plants, ammonia refrigeration systems, utilities, certain other manufacturers and wholesalers, and federal facilities. At press time, the rules were about to be published in the Federal Register. The rules list 139 regulated substances, with threshold reporting quantities ranging from 500 to 10,000 lb. There are significant differences between EPA's list of regulated chemicals and those in OSHA's process safety regulation. It is expected that these differences will be ironed out over the next three years. The chemical industry supports the regulations and has already made a good start toward meeting their requirements, says Matthew P. Weinstock, a spokesman for the Chemical Manufacturers Association. However, he notes, one of the tougher parts of complying will be presenting all the required infor-
