SCIENCE & TECHNOLOGY

HIV Viral core

Viral RNA

Cell Coreceptor membrane (CCR5or

CXCR4)

ATTACHMENT Binding of HIV's gp120 complex to CD4 and then a coreceptor— either CCR5 or CXCR4—causes gp41 to approach the cell surface and initiate the process of membrane fusion.

NIPPING AIDS IN THE BUD New class of medications prevents initial step in infection: Attachment to and entry into cells STU BORMAN, C&EN WASHINGTON

ALL COMMERCIAL DRUGS Di­rected specifically against the AIDS virus act by inhibiting either of two key human im­munodeficiency virus (HIV)

enzymes: reverse transcriptase or HlVpro-tease. The use of combinations, or "cock­tails," of these two classes of drugs has enabled a great number of HIV-infected individuals to keep the virus in check and stay alive.

But some patients don't respond to multidrug therapy; in many cases, side effects of current drugs are serious to severe; and the AIDS virus often develops resistance to existing therapies. Hence, there's a continuing need for new types of AIDS therapeutics.

One of the most promising avenues of investigation in the search for novel AIDS medications is the pursuit of viral entry inhibitors: drugs that prevent HIV from getting its proverbial foot in the door of target cells. Researchers in academia,

industry, and government are trying to identify and develop such drugs, and sev­eral of these groups discussed progress and prospects in this area last month at an American Chemical Society national meet­ing session on 'Antagonists of HI V Entry: Potential NewTherapies for Treatment of HIV Infection."

Reverse transcriptase inhibitors like Retrovir (zidovudine or AZT) interfere with conversion of viral RNA to DNA. Protease inhibitors like Viracept interfere with the maturation of the virus inside the cell. Entry inhibitors could be valuable additions to this AIDS drug armamentar­ium by blocking initial events in the infec­tion process: viral attachment to and entry into cells.

When a virus first encounters one of its cellular targets—a macrophage or Τ cell—a viral surface glycoprotein called gpl20 binds to CD4, a cell-surface recep­tor protein. This causes a conformational change ingpl20 that facilitates a second­

ary interaction: binding of gpl20 with a coreceptor.

TWO MAJOR CORECEPTORS used by HIV have been identified: CCR5 and CXCR4. Both are G-protein coupled receptors (GPCRs). "In the early stages of infection, the strains of HIV that pre­dominate use CD4 and CCR5 to access macrophages and T-cells," explains Jay Tagat, senior principal scientist at Scher­ing-Plough Research Institute. 'As infec­tion proceeds, other forms of HIV emerge, and they begin to utilize CD4 along with CXCR4. This often marks the transition from HIV infection to AIDS."

After attachment takes place, gp41, another component of the viral envelope complex, moves toward the target cell membrane, and a fusion peptide at the amino terminus of gp41 penetrates the membrane. Fusion, or melding of the viral and cell membranes, follows soon there­after, and the target cell becomes infected.

There are a number of ways to interfere with the overall process of viral cell entry One can try to block gpl20-CD4 inter­actions, gpl20-coreceptor binding, or gp41 interactions involved in membrane fusion. And researchers are trying all of them. For example, Progenies Pharmaceuticals recently completed Phase I and II clinical trials of PRO-542, an agent that binds gpl20 and prevents its attachment to CD4.

At the ACS symposium, researchers at Schering-Plough described work on a promising CCR5 antagonist, SCH-351125 (sometimes called SCH-Q. 'This is the first compound of its kind—a small-molecule CCR5 antagonist—to go into extensive clinical trials," says John Clader, distin­guished research fellow at Schering-Plough Research Institute. Clader chaired the symposium.

"There are no CCR5 inhibitory drugs out there now," Clader says. "So SCH-351125 would represent a new class of anti-HIV agents." Other companies, such as Merck, are also believed to be pursuing CCR5 antagonists.

THE CCR5 ANTAGONISTTAK-779 devel­oped by researchers at Takeda Chemical Industries, Osaka, Japan, was actually the first low-molecular-weight compound found to inhibit CCR5-based HIV entry [Proc. Natl. Acad. Sa. USA, 96, 5698 (1999)}. But TAK-779 isn't orally active and is generally administered by subcuta­neous injection. The drug's development as an injectable formulation was discon­tinued because of this difficulty "We are

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now investigating orally available CCR5 antagonists," Takeda research head Ύίιμ Iizawa tells C&EN.

SCH-351125 was discovered in Scher­ing-Plough's chemical research division by principal scientist Anan-dan Palani, working in Clader's group, and Tagat, working in the group of senior distin­guished research fellow Stuart McCombie. The discovery process was fast, taking only about two years in all.

By using high-throughput screening of Schering-Plough's com­pound library, Tagat and coworkers initially iden­tified several series of compounds that exhib­ited potent inhibition of the human CCR5 recep-tor. "We followed up on two series of com­pounds, " Tagat says. "One was based on a chi-ral piperazine core, and the other was based on a piperidine core. Our group worked on the chi-ral piperazine series," and another team led by Palani studied the piperidines.

high-throughput synthesis. "We had to optimize them for oral activity,"Tagat says. "We were trying to design compounds to be orally active so they could be adminis­tered as tablets or capsules."

THE COMPOUNDS were optimized by a combi­nation of traditional medicinal chemistry and

TARGET Model of CCR5 cell-surface receptor is shown from within plane of the cell membrane (left) and from above (right). CCR5 is a G-protein coupled receptor with seven transmembrane helices (cyan ribbons). In a study carried out last year, Dragic, Moore, Sakmar, and coworkers mutagenized the amino acid residues shown in space­filling representations. Residues were substituted with alanine to determine effects on inhibitor activity. The data suggested that the CCR5 inhibitor TAK-779 binds red amino acids primarily and orange and yellow residues to a lesser extent. Mutagenesis of dark blue residues had no substantial effect on binding, and mutagenesis of light blue residues caused CCR5 expression problems and couldn't be tested. Sakmar notes that the method only points to "the site where mutations prevent the ability of TAK-779 to inhibit HIV binding. This functional binding site must be related to the actual binding site— the sites on the receptor that make physical contact with bound TAK-779—and they might not be identical."

Based on several fac­tors, they eventually picked SCH-351125 from the piperidine series to be advanced fur­ther. It was taken into animal trials, where it exhibited good oral bioavailability in rats, dogs, and monkeys. It then entered human Phase I trials in Europe. Those tests are nearly complete, but results

from the trials aren't publicly available yet. Clader points out that "there's a natural

population of people who are resistant to HIV infection by virtue of the fact that they have a defect in their CCR5 receptor.

< These people can be => exposed to HIV, and S they don't get infected at | all." They appear to be S totally normal, healthy | individuals. So inhibiting § normal CCR5 receptors Ï with a drug like SCH-| 351125 is not expected to « cause serious side effects. | "That's the type of 0 thing that has really | encouraged people to

look at CCR5 antago­nists," Clader states. "We've got good reason to believe they will work." McCombie adds that Schering-Plough is also "currendy looking at candidates from a num­ber of chemical series as potential follow-ups" to SCH-351125.

Also discussed at the ACS symposium was a fundamental study of CCR5 receptor binding carried out recently by Tatjana Dragic, now at the department of mi­crobiology and immu­nology of Albert Einstein College of Medicine; professor John R Moore, now at the department

One of the most promising avenues of investigation in the search for novel AIDS medications is the pursuit of viral entry inhibitors.

H T T P : / / P U B S . A C S . O R G / C E N C&EN / MAY 2 1 . 2001 65

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of microbiology and immunology at Weill Medical College of Cornell University; Thomas P. Sakmar of Howard Hughes Medical Institute and the laboratory of molecular biology and biochemistry at Rockefeller University; and coworkers [PrOc.Natl.Acad.Sci. USA, 97,5639(2000)}.

THE PURPOSE of the study was to map small-molecule binding domains on CCR5, Sakmar explained. This was done by using site-directed mutagenesis to look at muta­tions that prevent the receptor from being a drug target of a small molecule.

"We looked for mutants that affected the ability ofTAK-779 to bind to the recep-tor," Sakmar says. "Then we mapped the locations of the mutants onto a hypothet­ical model of the receptor and tried to determine where the small molecule binds—with the idea that from this you could get some insights into designing bet­ter or more specific molecules.,,

TAK-779, and by analogy probably other CCR5 inhibitors like SCH-351125, interact with the seven-helix transmem­brane domain of the CCR5 coreceptor. ThePNAS study suggested thatTAK-779 binds primarily within a cavity between transmembrane helices 1,2,3, and 7, close to the receptor's extracellular surface boundary teWc intend to use the same strat­egy to map the binding sites of other of the small molecules that have anti-HIV activ­ity," Sakmar says.

Thanks to Sakmar and coworkers, "we're beginning to get a picture of what the CCR5 receptor might look like and beginning to make at least some specula­tions about how compounds are interact­ing with the CCR5 receptor," Clader says. 'That's the type of thing that is really going to facilitate research in this area."

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BICYCLAMS De Clercq and coworkers discovered the CXCR4 inhibitors AMD-3100 and AMD-7049.

Meanwhile, professor Erik De Clercq and coworkers at the Rega Institute for Medical Research of Catholic University, Leuven, Belgium, in collaboration with researchers at AnorMED, Langley, British Columbia, have discovered that a bicyclam compound called AMD-3100 and several analogs help fight HIV infection by potently inhibiting CXCR4 coreceptor interactions. AMD-3100 is a highly spe­cific antagonist of CXCR4, in that it blocks signal transduction only through this receptor and not through any of sev­eral other receptors.

IN VITRO and animal tests carried out by the researchers show that AMD-3100 con­sistently blocks the replication of HIV variants that use CXCR4 for entering T-cells. The compound passed Phase I trials in 1999 and is now undergoing Phase II trials in HIV-infected subjects.

The compound has limited oral bioavail­ability, but De Clercq and coworkers hope this problem can be remedied with analogs. The researchers recently synthesized AMD-7049, an analog that acts as a CXCR4 antagonist and blocks HIV infec­tion with a potency similar to that of AMD-3100 but is orally bioavailable. They've also identified an analog called AMD-3451 that interacts with both CCR5 and CXCR4 and inhibits the replication of HIV strains that use either of the two receptors for cell entry

Researchers at Trimeris are pursuing drugs to inhibit yet another phase of HI V entry: viral fusion, the final step in the entry process. After HIV's gpl20 com­plex binds to the CD4 receptor and either the CCR5 or CXCR4 coreceptor, the virus's gp41 component injects fusion pep­tides into the cell membrane, anchoring

gp41 to the membrane. Two domains of gp41—heptad repeat-1 and -2 (HR-1 and HR-2)- then fold together in a process called HR-2 zipping to form a six-heli­cal bundle structure. This brings the virus and target cell surfaces closer together, resulting eventu­ally in membrane fusion.

At the ACS meeting, Trimeris senior vice president of develop­ment M. C. Kang discussed T20 andT-1249, two linear synthetic peptides that have sequences mimicking that of HR-2. The two HR-2 mimics interact with a key hydrophobic groove on the HR-1 trimer core, which prevents the contiguous viral HR-2 domain from zipping up with HR-1. This

66 C&EN / MAY 2 1 . 2001 HTTP: / / P U B S . ACS.ORG/CEN

Viral membrane

Target cell

membrane

Receptor Fusion binding —• intermediate - • HR-2 zipping -

Six-helix • bundle formation

FUSION Following binding of the viral envelope glycoprotein gp120 (not shown) with cellular CD4 and coreceptors, structural changes in gp120 are believed to release hydrophobic fusion peptides at the amino terminus of gp41 (arrows penetrating cellular membrane). The changes in gp120 also expose two gp41 heptad repeat domains, HR-1 and HR-2. Folding of HR-2 against HR-1's trimeric core forms a six-helix bundle and acts to draw the viral and cellular membranes closer together, leading to membrane fusion. T-20, a compound whose sequence mimics that of HR-2, binds to and occupies grooves on the HR-1 trimeric core, blocking six-helix bundle formation and membrane fusion.

blocks formation of the gp41 six-helix bun­dle and inhibits viral fusion.

According to Kang, T-20 is the first fusion inhibitor to show antiviral activity in people. It inhibits fusion at nanogram-per-milliliter levels in vitro "and has achieved proof of concept as a potent inhibitor of HIV replication in humans," he says. It's currently in Phase III clinical trials, and its analogT-1249 is undergoing Phase I trials. Hoffmann-La Roche is code-veloping the two agents withTrimeris.

T-20 AND T-1249 currently require non-oral administration. 'At the moment, this is a twice-a-day subcutaneous injection," Kang says. But he notes thatTHmeris and Roche are trying to develop improved dosage forms that will be more convenient to patients.

Kang says the drugs are potential com­plements to current AIDS cocktails and are also appropriate "for people resistant to conventional multidrug therapy. The viral count rebounds very rapidly once a patient begins to fail a drug regimen for the second time," he says. "In such a case, there are limited options to offer. The com­munity really needs a drug that works with a different mechanism to improve the resistance pattern."

Producing a peptide drug like T-20 on a commercial scale is not a trivial endeavor. The current dose is 100 to 200 mg per day— about 75 g per year per patient at the

highest dose. When you consider the large population of HIV-infected individuals and AIDS patients who could potentially benefit from the drug, "this is a large quan­tity of peptide," Kang says. "We believe we will need multiple metric tons of T20 at commercial scale. This will be the first pep­tide drug manufactured chemically on that scale."

A team of chemists at Trimeris, led by Brian Bray, "has developed a unique man­ufacturing process to make unprecedented amounts of this complex medicinal pep­tide," Kang says. "We are already making it in large quantities and we will eventually be able to make it on a metric-ton scale using mostly conventional equipment." He saysTrimeris hopes to file a new-drug appli­cation for T-20 with the Food & Drug Administration by next year.

Although significant progress has been made on the development of inhibitors targeted at CD4, CCR5, CXCR4, and gp41, "still more progress will be needed before any of these compounds makes it to the drug market," De Clercq says.

However, in the ongoing search for new AIDS therapies, "basic studies of the biol­ogy of HIV entry and targeted pharma­ceutical development are progressing in parallel," Sakmar tells C&EN. And it's becoming increasingly clear that the advent of a new class of HIV entry inhibitory drugs is no longer a matter of "if," but "when."·

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