Adaptive licensing: taking the next step in the evolution of drug approval.

Traditional drug licensing approaches are based on binary decisions. At the moment of licensing, an experimental therapy is presumptively transformed into a fully vetted, safe, efficacious therapy. By contrast, adaptive licensing (AL) approaches are based on stepwise learning under conditions of acknowledged uncertainty, with iterative phases of data gathering and regulatory evaluation. This approach allows approval to align more closely with patient needs for timely access to new technologies and for data to inform medical decisions. The concept of AL embraces a range of perspectives. Some see AL as an evolutionary step, extending elements that are now in place. Others envision a transformative framework that may require legislative action before implementation. This article summarizes recent AL proposals; discusses how proposals might be translated into practice, with illustrations in different therapeutic areas; and identifies unresolved issues to inform decisions on the design and implementation of AL.

Crystallization and preliminary X-ray study of the edema factor exotoxin adenylyl cyclase domain from Bacillus anthracis in the presence of its activator, calmodulin.

Edema factor from Bacillus anthracis is a 92 kDa secreted adenylyl cyclase exotoxin and is activated by the host-resident protein calmodulin. Calmodulin is a ubiquitous intracellular calcium sensor in eukaryotes and activates edema factor nearly 1000-fold upon binding. While calmodulin has many known effectors, including kinases, phosphodiesterases, motor proteins, channels and type 1 adenylyl cyclases, no structures of calmodulin in complex with a functional enzyme have been solved. The crystallization and initial experimental phasing of crystals containing a complex of edema factor adenylyl cyclase domain and calmodulin are reported here. The edema factor-calmodulin complex crystallizes in three different space groups. A native data set in the I222 space group has been collected to 2.7 A and the self-rotation function solution suggests three edema factor-calmodulin complexes in each asymmetric unit. Initial 4 A phases were obtained by selenomethionyl MAD in combination with two heavy-atom derivatives. These phases were successfully extended to 2.7 A using NCS averaging.

An extended conformation of calmodulin induces interactions between the structural domains of adenylyl cyclase from Bacillus anthracis to promote catalysis.

The edema factor exotoxin produced by Bacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed in Escherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis and Pseudomonas aeruginosa, is highly resistant to protease digestion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM-dependent protein kinase type II. Mutations in each of the four calcium binding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic screen probing loss-of-function mutations of EF3 and site-directed mutations based on the homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.

Differences in the binding sites of two site-3 sodium channel toxins.

Site-3 toxins from scorpion and sea anemone bind to Na channels and selectively inhibit current decay. Anthopleurins A and B (ApA and ApB, respectively), toxins found in the venom of the sea anemone Anthopleura xanthogrammica, bind to closed states of mammalian skeletal and cardiac Na channels with differing affinities which arise from differences in first-order toxin/channel dissociation rate constants, koff. Using chimera comprising domain interchanges between channel isoforms, we examined the structural basis of this differential affinity. Toxin/channel association rates, kon, were similar for both toxins and both parental channels. Domain 4 determined koff for ApA, while ApB dissociated from all tested chimera in a cardiac-like manner. To probe this surprising difference between two such closely related toxins, we examined the interaction of chimeric channels with a form of ApB in which the two nonconserved basic residues, Arg-12 and Lys-49, were converted to the corresponding neutral amino acids from ApA. In the chimera comprising domain 1 from the cardiac muscle isoform and domains 2-4 from the skeletal muscle isoform, toxin dissociated at a rate intermediate between those of the parental channels. We conclude that the differential component of ApA binding is controlled by domain 4 and that some component of ApB binding is not shared by ApA. This additional component probably binds to an interface between channel domains and is partly mediated by toxin residues Arg-12 and Lys-49.

Role for Pro-13 in directing high-affinity binding of anthopleurin B to the voltage-sensitive sodium channel.

Anthopleurin A (ApA) and B (ApB) are 49-amino acid polypeptide toxins from the Pacific sea anemone Anthopleura xanthogrammica that interfere with inactivation of voltage-gated sodium channels. ApA, which differs from ApB in seven of the 49 amino acids, displays markedly enhanced isoform selectivity compared with ApB, acting preferentially on cardiac over neuronal sodium channels. Previous studies in this lab have indicated the importance of two unique charged residues in ApB, Arg-12 and Lys-49, in this toxin's ability to discriminate between neuronal and cardiac sodium channels. Likewise, a double mutant (R12S/K49Q) recently characterized in this lab (Khera et al., 1995) displays a greatly reduced affinity for neuronal channels, essentially restoring the discriminatory ability of ApA. When the remaining five residues unique to ApB are individually converted to those of ApA, only ApB (Pro-13) shows a major effect, reducing the affinity of the new mutant toxin (P13V) against both channel isoforms approximately 10-fold. This effect is most likely the result of a conformational rearrangement within the surrounding cationic cluster which includes Arg-12 and -14, as well as Lys-49. However, when placed into the context of the double mutant R12S/K49Q a unique effect is observed: the new triple mutant (R12S/P13V/K49Q) is no longer able to discriminate effectively between channel isoforms. Its affinity for the neuronal sodium channel is significantly enhanced compared to either P13V or to the double mutant R12S/K49Q. These results are consistent both with our proposed model (Khera et al., 1995) and with the recently reported solution structure of ApB, which implicate the cationic cluster in both affinity and channel isoform selectivity. We suggest that the P13V mutation results in a shift in the relative orientation of cationic residues within the large flexible loop between residues 9-18, thus strengthening their interactions with target sequences of the neuronal sodium channel.

The role of exposed tryptophan residues in the activity of the cardiotonic polypeptide anthopleurin B.

Scorpion and sea anemone venoms contain several polypeptides that delay inactivation of voltage-sensitive sodium channels via interaction with a common site. In this report, we target exposed hydrophobic residues at positions 33 and 45 of anthopleurin B (ApB) by polymerase chain reaction mutagenesis to ascertain their contribution to toxin activity. Nonconservative replacements are not permitted at position 33, indicating that Trp-33 may play an important structural role. Strikingly, the relatively conservative substitution of Trp-33 by phenylalanine results in major reductions in binding affinity for both the cardiac and neuronal channel isoforms as measured by ion flux, whereas substitution with tyrosine is tolerated and exhibits near wild-type affinities, suggesting that either the ability to form a hydrogen bond or the amphiphilic nature of the side chain are important at this position. Electrophysiological analysis of W33F indicates that its diminished affinity is primarily due to a decreased association rate. Analysis of a panel of mutants at Trp-45 shows only modest changes in apparent binding affinity for both channel isoforms but significant effects on Vmax. In neuronal channels, the maximal levels of uptake for W45A/S/F are about 50% those seen with ApB. This effect is also observed for W45A and W45S in the cardiac model, wherein W45F is normal. These results suggest that a hydrophobic contact is involved in toxin-induced stabilization of the open conformation of the cardiac sodium channel. We conclude that Trp-33 contributes significantly to apparent affinity, whereas Trp-45 does not appear to affect binding per se. Furthermore, W33F is the first ApB mutant that displays a significantly altered association rate and may prove to be a useful probe of the channel binding site.

Leucine 18, a hydrophobic residue essential for high affinity binding of anthopleurin B to the voltage-sensitive sodium channel.

Anthopleurin B is a potent anemone toxin that binds with nanomolar affinity to the cardiac and neuronal isoforms of the voltage-gated sodium channel. A cationic cluster that includes Arg-12, Arg-14 and Lys-49 has been shown previously to be important in this interaction. In this study, we have used site-directed mutagenesis to determine the contribution to activity of two aliphatic residues, Leu-18 and Ile-43, that have previously been experimentally inaccessible. Leu-18, a residue proximal to the cationic cluster, plays a critical role in defining the high affinity of the toxin. In ion flux studies, this is exemplified by the several hundredfold loss in affinity (231-672-fold) observed for both L18A and L18V toxins on either isoform of the sodium channel. When analyzed electrophysiologically, L18A, the most severely compromised mutant, also displays a substantial loss in affinity (34-fold and 328-fold) for the neuronal and cardiac isoforms. This difference in affinities may reflect an increased preference of the L18A mutant for the closed state of the neuronal channel. In contrast, Ile-43, a residue distal to the cationic cluster, plays at most a very modest role in affinity toward both isoforms of the sodium channel. Only conservative substitutions are tolerated at this position, implying that it may contribute to an important structural component. Our results indicate that Leu-18 is the most significant single contributor to the high affinity of Anthopleurin B identified to date. These results have extended the binding site beyond the cationic cluster to include Leu-18 and broadened our emphasis from the basic residues to include the crucial role of hydrophobic residues in toxin-receptor interactions.

Multiple cationic residues of anthopleurin B that determine high affinity and channel isoform discrimination.

Site 3 sea anemone toxins modify inactivation of mammalian voltage-gated Na channels. One variant, anthopleurin A (ApA), effectively selects for cardiac over neuronal mammalian isoforms while another, anthopleurin B (ApB), which differs in 7 of 49 amino acids, modifies both cardiac and neuronal channels with high and approximately equal affinity. Previous investigations have suggested an important role for cationic residues in determination of toxin activity, and our single-site mutagenesis studies have indicated that isoform discrimination can be partially explained by the unique cationic residues Arg-12 and Lys-49 of anthopleurin B (ApB). Here, we have further investigated the role of cationic residues by characterizing toxin mutants in which two such residues are replaced simultaneously. The ApB double mutants R14Q-K48A (cationic residues identical in both ApA and ApB), R12S-K49Q (cationic residues unique to ApB), and R12S-R14Q (cationic residues located in the unstructured loop shared among anemone toxins) were constructed by site-directed mutagenesis and their biological activities characterized by sodium uptake assays in cell lines expressing the neuronal (N1E-115) or cardiac (RT4-B) isoform of the Na channel. Each double mutant displayed reduced activity compared with wild type, but none were completely inactive. Neutralization of the proximal cationic residues (R12 and R14) was the most effective, reducing affinity 72-fold (neuronal) and 56-fold (cardiac). Substitution of cationic residues that differed between ApB and ApA (R12S-K49Q) reduced affinity of the toxin for neuronal channels to a much greater extent than for cardiac channels, producing affinities only slightly lower than for ApA in each case.(ABSTRACT TRUNCATED AT 250 WORDS)