ST-246 is a low-molecular-weight compound (molecular weight‫؍‬Recent concerns over the use of variola (smallpox) virus as a biological weapon have prompted renewed interest in development of small molecule therapeutics that target variola virus replication. Currently, there is no U.S. Food and Drug Administration-approved drug for the prevention or treatment of smallpox infection. While a number of compounds have been shown to inhibit orthopoxvirus replication in vitro, these compounds often lack potency and/or are associated with significant adverse effects, due to their relative nonspecific mechanisms of virus inhibition (3).The cornerstone of the current national public health response plan to a smallpox bioterrorist attack calls for rapid mass immunization with vaccinia virus. However, concerns about vaccine-related adverse events have compromised implementation of a smallpox immunization program. Individuals with immunodeficiency disorders or certain common skin conditions are unusually susceptible to vaccine-related complications (6, 32). Moreover, the lag period for antibody formation from a vaccine leaves a window of vulnerability. Antiviral therapies can fill this void and provide an excellent complement to vaccination in that they reduce virus titers quickly, regardless of immune status, and lower transmission rates by diminishing the virus reservoir. A small-molecule antiviral drug designed to treat variola virus infection will be a critical component to a smallpox defense strategy.Currently, only cidofovir [CDV; (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine; Vistide], a drug approved for treatment of cytomegalovirus (CMV) retinitis in AIDS pa-* Corresponding author. Mailing address:

Group A streptococci are human pathogens that infect primarily at the skin or nasopharyngeal mucosa . A major virulence factor present on the streptococcal surface is M protein, a molecule of which there exists more than 80 distinct serological types. M protein provides the streptococcus with the ability to resist phagocytosis by polymorphonuclear leukocytes, and only antibodies directed to type-specific determinants permit opsonophagocytosis in whole blood (1, 2). Structural studies reveal that M protein is composed of two a-helical polypeptide chains that give rise to a coiled-coil, fibrillar structure extending -60 nm from the cell surface (3, 4). The antigenically variable determinants of type specificity are located in the NH2-terminal portion of the molecule, distal to the cell wall (2, 5). Some epitopes of the Mprotein molecule are shared among different M serotypes, and the degree ofrelatedness increases at sites closer to the COOH terminus (6, 7).Recent progress towards a group A streptococcal vaccine indicates that mucosal immunization with shared immunodeterminants of M protein leads to reduced pharyngeal colonization in a mouse model (8). To better define those epitopes for potential use in a non-type-specific-based vaccine, we sought to gain a more detailed understanding of the antigenic relatedness of surface-exposed portions of M protein among isolates of the same serotype, and between isolates of distinct serotypes. In this report, we describe an antigenically conserved domain within the surface-exposed portion of M protein of certain serological types, and provide evidence for two distinct classes of M protein molecules based on the presence or absence of this antigenic domain.Acute rheumatic fever (ARF)' is one complication that can follow a nasopharyngeal streptococcal infection, and epidemiological studies point to a strong association between streptococci ofcertain serological M types and ARF attacks (9). Defining a particular serotype as "rheumatogenic" is not entirely clearcut since pharyngitis generally precedes the onset of ARF by 3-4 wk and thus, there is uncertainty as to whether the streptococcus isolated at the time of an ARF attack is of the same lineage as that which caused pharyngitis. Nevertheless, it is apparent that streptococci

The M protein of group A streptococci is a fibrous dimer of a helices arranged in a coiled coil extending^" 50 nm from the surface of these organisms (1, 2) .Due to its profound antiphagocytic effect, this molecule serves as the primary virulence factor for these streptococci (3) which are cleared from the infected host only after type-specific opsonic antibodies are produced to the M protein (4). mAbs to the native M6 protein and DNA hybridization studies on the M6 gene indicate that the N11 2 -terminal region of this molecule is variable among M serotypes, whereas the carboxy-terminal half is conserved (5-8) . This suggests that type specificity may reside in the NH2-terminal half of the M protein, which is distal to the cell surface (9) . While a number of studies on a variety of M protein fragments derived from the NH 2-terminal half of the molecule suggest the importance of this region in the production of opsonic antibodies (10-14), no systematic approach concerning the binding site and function of specific antibodies to the complete M molecule has yet been reported .To help delineate the location of opsonogenic determinants on the M6 protein, mAbs raised against the native M6 molecule were examined for their ability to opsonize type 6 streptococci . Four of these monoclonals that had defined epitopes (reference 6 ; Jones, K . F ., S . K . Hollingshead, J . R . Scott, and V . A . Fischetti, manuscript in preparation) were examined for their IgG subclasses and ability to fix human complement . In addition, antisera against synthetic peptides representing various areas of the native M6 protein were examined for their opsonic capabilities . These studies revealed that opsonization of streptococci is a function of the location of the antibody-binding site on the M molecule . Materials and MethodsPurified M6 Proteins. M6 protein extracted from streptococcal cells by group C streptococcal phage-associated lysin (LysM6) was purified as described (15). M6 protein (ColiM6) was extracted and purified from an Escherichia coli strain expressing the structural gene for M6 protein (9) .Production of mAbs. mAbs against either LysM6 or ColiM6 were produced and protein A purified from culture supernatants as described (5, 6) .Bacterial Strains. Group A streptococcal clinical isolates representing 56 M serotypes were from The Rockefeller University culture collection and were listed previously (6) .This research was supported by a Public Health Service grant to V. A. Fischetti (AI-11822) . 1114J. Exp. Men.

Category A arenaviruses as defined by the National Institute of Allergy and Infectious Diseases (NIAID) are human pathogens that could be weaponized by bioterrorists. Many of these deadly viruses require biosafety level-4 (BSL-4) containment for all laboratory work, which limits traditional laboratory high-throughput screening (HTS) for identification of small molecule inhibitors. For those reasons, a related BSL-2 New World arenavirus, Tacaribe virus, 67-78% identical to Junín virus at the amino acid level, was used in a HTS campaign where approximately 400,000 small molecule compounds were screened in a Tacaribe virus-induced cytopathic effect (CPE) assay. Compounds identified in this screen showed antiviral activity and specificity against not only Tacaribe virus, but also the Category A New World arenaviruses (Junín, Machupo, and Guanarito). Drug resistant variants were isolated, suggesting that these compounds act through inhibition of a viral protein, the viral glycoprotein (GP2), and not through cellular toxicity mechanisms. A lead compound, ST-294, has been chosen for drug development. This potent and selective compound, with good bioavailability, demonstrated protective anti-viral efficacy in a Tacaribe mouse challenge model. This series of compounds represent a new class of inhibitors that may warrant further development for potential inclusion in a strategic stockpile.

The overexpression of multidrug transporters significantly reduces the accumulation of protease inhibitors at this major site of virus replication, which, potentially, could accelerate the acquisition of viral resistance. Targeted inhibition of P-gp may represent an important strategy by which this problem can be overcome.

No abstract

M protein is a fibrillar molecule composed of two alpha-helical protein chains in a coiled-coil conformation extending from the surface of group A streptococci (1, 2). This molecule is the major virulence factor for the organism by virtue of its ability to render the bacterium resistant to phagocytosis in the human host (3). Based on type-specific antigenic determinants on the M molecule, approximately 65 different serological M types are currently recognized in nature. In man, resistance to streptococcal infection appears to be the result of the presence of type-specific antibodies directed to the M molecule which will neutralize its antiphagocytic properties (3).Previous investigations, which revealed very limited immunological crossreactions between purified M proteins of different serotypes, were primarily confined to detergent-extracted (4) or pepsin-extracted (5) M protein fragments. These fragments comprise only the amino-terminal half of the M molecule (2, 6) and, thus, do not represent all of the determinants present on the native protein.Development of a vaccine to control streptococcal infections may depend upon the identification of a common M protein epitope with the ability to evoke crossprotective antibodies against multiple group A streptococcal serotypes. Since 56 different M serotyping strains show DNA homology with a probe consisting of the gene for the M6 molecule, a common region(s) may exist within the M proteins of different serotypes (7).In an attempt to identify the location of epitopes responsible for broad and limited crossreactivity among 56 of the currently known group A streptococcal M serotypes, we have used monoclonal antibodies generated against phage lysinextracted M6 protein, which comprises nearly the complete M molecule (8). Our results localize a common (evolutionarily conserved) crossreactive epitope near This work was supported by grants AI 11822 (V. Fischetti) and

Streptococcal M protein, the fibrillar molecule on the surface of the group A streptococcus, is considered to be one of the major factors responsible for the ability of this human pathogen to cause disease (1). Though type-specific opsonic antibodies directed towards the M molecule enable the infected host to clear the invading streptococcus (1, 2), the ability of this organism to vary its antigenic structure has probably been the major factor responsible for its survival. To date, over 75 serologically different M proteins have been identified in nature along with an increasing number of nontypable strains isolated from infected individuals (World Health Organization Conference, 1984, Tokyo, Japan).Based on structural and physicochemical studies, it appears that the M protein is composed of two predominantly alpha-helical protein chains assembled in a coiled-coil (3-5) and extending nearly 600 A. from the cell surface (5). Partial sequence analysis of three different M molecules has revealed that these sequences are essentially different, but have a common placement of hydrophobic residues (4, 6, 7) that is responsible for maintaining the coiled-coil structure (4, 5) common to those M molecules studied. Though many of these studies have been useful in understanding the structure and immunochemistry of the M molecule, they have been primarily confined to an amino-terminal fragment of the protein comprising nearly half the molecule (pepsin-extracted M protein) (4, 6, 7).Several different procedures for obtaining M protein from whole streptococci or their isolated cell walls have been used, including extraction with acid (8), alkali (9), pepsin (10, 11), and nonionic detergent (12). In all cases, however, the resulting product was probably a fragment of the native molecule. Despite some attempts to isolate the M protein from the streptococcus with cell wail-solubilizing enzymes (13-16), the complexity of the resulting digest has offered little information about the nature of the native molecule as it exists on the streptococcus. To avoid the destructive properties of certain extraction techniques, M protein was purified from the secreted products of streptococcal L forms and protoplasts of type 12 streptococci (17). The molecular weight of the M protein monomer from these two sources was 58,000. In more recent studies, Scott and Fischetti (18) cloned the gene for M6 in Escherichia coli, and Fischetti et al. (19)