Picture an ordinary wooden barrel, composed of seven staves. Nowimagine this three- or four-foot-high container shrunk down 10billion times to the length of an angstrom.
That's about the diameter of a single atom. It's the unit ofmeasurement that protein crystallographers use to solve the 3-Dstructures of their molecules.
Nature, in its current issue, dated Sept. 19, 1996, carries threeremarkably homologous papers reporting the structure of the sameserine protease enzyme, which enables the cytomegalovirus toreplicate. To wit:
* "Three-dimensional structure of human cytomegalovirus protease,"out of Monsanto Co. / G.D. Searle & Co. in Skokie, Ill.;
* "Unique fold and active site in cytomegalovirus protease," fromSmithKline Beecham Pharmaceuticals in King of Prussia, Pa.
* "A new serine-protease fold revealed by the crystal structure ofhuman cytomegalovirus protease," by Boehringer-IngelheimPharmaceuticals Inc., in Ridgefield, Conn.
Aside from the slight variations in their titles, and the fact that thefour companies solved the structures to resolutions of 2.27, 3.0 and2.0 angstroms respectively, "there are no profound differences amongthe three papers," observed molecular virologist Barry Holwerda,who headed the Monsanto/Searle project, "so basically one papercould have done."
Holwerda added that besides the work just published by these threegroups, "We think there are others as yet unannounced."
The glittering prize they all seek is a drug to inhibit the serineprotease in cytomegalovirus (CMV), which has recently emerged as alife-threatening scourge.
"It causes disease that we don't see unless an individual becomesimmunocompromised," Holwerda told BioWorld Today, "so theaffected populations are basically AIDS patients, newborns, anybodyundergoing chemical immunosuppression, especially for bonemarrow transplantation."
Subclinical In Most; Lethal In Some
CMV, a member of the herpesvirus family, is widely prevalent. Bythe age of about 60 to 70, some 70 percent of the population has beeninfected, but its symptoms are generally subclinical.
"For late-stage AIDS patients," Holwerda observed, "the mostprofound symptom that CMV causes is retinitis, which pretty muchleads to blindness in about 20 percent of them." (Chiron Corp., ofEmeryville, Calif., is launching an ocular implant that delivers anantiviral drug to the retina. See BioWorld Today, March 6, 1996, p.2.)
He added: "The other problem with the AIDS population is that anawful lot of patients die from a disseminated CMV infectionthroughout most of their organ systems. For an immunosuppressedbone marrow transplant patient, we see severe pneumonitis, possiblerenal failure. Hepatitis is a possible outcome, and in some casesencephalitis. CMV can infect the central nervous system."
A prime target of drug discovery is CMV's 256-amino-acid serineprotease enzyme, which is the sine qua non of its replication in aninfected cell.
"You can knock this enzyme out genetically," Holwerda pointed out,"and you get a big loss _ five to 10 orders of magnitude _ in viralyield. It's been done in CMV's cousin, herpes simplex virus type one_ the cold-sore virus. It showed that if you knock the serine proteaseout, the virus cannot grow."
"It's generally believed," added Holwerda's associate, medicinal andstructural chemist William Stallings, "that the herpesvirus forms anassembly structural protein as a template for the virion capsid. Beforethe viral DNA can be packaged inside the virus, it has to get rid ofthat scaffolding, which is what the serine protease does."
Stallings, who heads Monsanto/Searle's research crystallographygroup, is his paper's senior author. All three Nature articles describethe same structural folding of the CMV enzyme.
"That fold," Stallings told BioWorld Today, "is a polypeptide chain.More than half of it folds into a barrel-shaped configuration withseven staves, which we call strands or beta sheets. The strands at theend of this central feature lead out into a helical structure, whichcompletely encircles the barrel. It's the first such fold that's beendetermined."
He continued: "Aligning the amino acid sequences of this one withother herpes proteases, suggested to us that they are all going to foldlike that. Actually, the parts that protrude from the top and bottom ofthe barrel might make one herpes protease different from another."
Beyond Folding There's Dimerization
"To be active," Stallings continued, "the molecule needs twopolypeptide chains to form a dimer complex."
"We characterized those folds and dimerization only for CMV,"Holwerda observed, "but I'll say this: There are several elements ofsecondary structure in that crystal that are conserved among all theherpesvirus proteases, so we could predict that the dimerizationphenomenon would be common to all of them."
The industry rush is on to find drugs that can prevent thisdimerization.
"The approach," Stallings said, "is a lot like that from HIV protease,which is all over the news. Academics in particular," he added, "arethinking that if you can block dimerization of the enzyme, that wouldbe a way of inhibiting it."
"And one of the big challenges for everyone working on this,"Holwerda emphasized, "is that such inhibitors must be specific just tothe viral serine protease.
"This obligate specificity," he explained, "reflects the fact that all theblood coagulation cascade enzymes _ thrombin, kallikrein, FactorXa _ are serine proteases."
But he also pointed out, "Although this is a serine proteinase, there isvery little resemblance in terms of its activity to such serine proteasesin the human body as chymotrypsin, [a digestive enzyme in the gut]human leukocyte elastase, [which cleaves elastin in connectivetissues], even, for that matter, subtilisin [another digestiveproteinase]."
Holwerda concluded: "The industry is moving toward an orallyavailable inhibitor, so that the patient just takes a pill. This wouldmake things a lot easier, especially for AIDS patients." n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.