The association of a drug with its target protein has the effect of blocking the protein activity and is termed a promiscuous function to distinguish from the protein's native function (Tawfik and associates, Alat. Genet. 37,73-6,2005). Obviously, a protein has not evolved naturally for drug association or drug resistance. Promiscuous protein functions exhibit unique traits of evolutionary adaptability, or evolvability, which is dependent on the induction of novel phenotypic traits by a small number of mutations. These mutations might have small effects on native functions, but large effects on promiscuous function; for example, an evolving protein could become increasingly drug resistant while maintaining its original function. Ariel Fernandez, in his opinion piece, notes that drug-binding "promiscuity" can hardly be dissociated from native functions; a dominant approach to drug discovery is the protein-native-substrate transition-state mimetic strategy. Thus, man-made ligands (e.g. drugs) have been successfully crafted to restrain enzymatic activity by focusing on the very same structural features that determine the native function. Using the successful inhibition of HIV-1 protease as an example, Fernandez illustrates how drug designers have employed naturally evolved features of the protein to suppress its activity. Based on these arguments, he dismisses the notion that drug binding is quintessentially promiscuous, even though in principle, proteins did not evolve to associate with man made ligands. In short, Fernandez argues that there may not be separate protein domains that one could term promiscuous domains. While acknowledging that drugs may bind promiscuously or in a native-like manner a la Fernandez, Tawfik maintains the role of evolutionary adaptation, even when a drug binds native-like. In the case of HIV-1 protease, drugs bind natively, and the initial onset of mutations results in drug resistance in addition to a dramatic decline in enzymatic activity and fitness of the virus. A chain of compensatory mutations follows this, and then the virus becomes fully fit and drug resistant. Ben Berkhout and Rogier Sanders subscribe to the evolution of new protein functions through gene duplication. With two identical protein domains, one domain can be released from a constraint imposed by the original function and it is thus free to move in sequence space toward a new function without loss of the original function. They emphasize that the forced evolution of drug-resistance differs significantly from the spontaneous evolution of an additional protein function. For instance, the latter process could proceed gradually on an evolutionary time scale, whereas the acquisition of drug-resistance is an all or nothing process for a virus, leading to the failure or success of therapy. They find no evidence to the thesis that resistance-mutations appear more rapidly in promiscuous domains than native domains. Berkhout and Sanders illustrate the genetic plasticity of HIV-1 by citing examples in which well-conserved amino acid residues of catalytic domains are forced to mutate under drug-pressure. HIV drug resistance biology is very complex. Instead of a viral protein, a drug can be targeted at a cellular protein. For example, Berkhout and Sanders claim, a drug targeted at the cellular protein CCR5 inhibits the binding of the viral envelope glycoprotein (Env) to CCR5. However, Env mutates so that it binds to the CCR5-drug complex and develops drug resistance. Interestingly, CCR5 has not evolved to bind to Env, but to a series of chemokines. Andrzej Kloczkowski, Taner Sen, and Bob Jernigan point out the importance of protein motions for binding. They believe it is likely that different ligands can bind to the diverse protein conformations sampled in the course of normal protein conformational fluctuations. They have been applying simple elastic network models to extract the motions as normal modes, which yield relatively small numbers of conformations that are useful for developing protein mechanisms; while these are typically small motions, for some proteins they can be quite large in scale. One of the major advantages of the approach is that only relatively small numbers of modes are important contributors to the overall motion - so the approach provides a way to systematically map out a protein's motions. These models successfully represent the conformational fluctuations manifested in the crystallographic B-factors, and often suggest motions related to protein functional behaviors, such as those observed for reverse transcriptase, where two dominant hinges clearly relate to the processing steps - one showing anti-correlation between the polymerase and ribonuclease H sites related to the translation and positioning of the nucleic acid chain, and another for opening and closing the polymerase site. Disordered proteins represent a more extreme case where the set of accessible conformations is much larger; thus they could offer up a broader range of possible binding forms. Whether evolution controls the functional motions for proteins remains little studied. Intriguingly, buried in the existing databases of protein-protein interactions may be information that can shed light on the extent of promiscuous binding among proteins themselves. Within these data there are cases where large numbers of diverse proteins have been shown to interact with a single protein; some of these could represent promiscuous protein-protein binding. Uncovering these promiscuous behaviors could be important for comprehending the details of how proteins can bind promiscuously to one another, and can exhibit even greater promiscuity in their binding to small molecules. Most researchers assume a clear delineation between native and promiscuous protein functions (1). In contrast with native functions, promiscuous functions are assumed to involve predominantly entropy-driven interactions, to typically exclude pair-wise enthalpic contributions, and to be essentially free from selection pressure, in accord with their purported latency. Thus, a conspicuous illustration of promiscuity is assumed to be enzymatic inhibition by drug association (2 - 6), a function for which the protein clearly has not evolved naturally. I believe this view needs revision in the light of the following considerations. There are numerous instances where drug-binding promiscuity can hardly be dissociated from native function, as evidenced by the fact that a dominant approach to drug discovery is the protein-native-substrate transition-state mimetic strategy (26). Thus, man-made ligands, for example drug inhibitors, have been successfully made to inhibit enzymatic activity by focusing on the very same structural features that determined the native function. Furthermore, there are native structural features germane to enzymatic processivity which have been obviously subject to severe selection pressure for a particular role and are utilized in an alternative role in what Aharoni et al. (1) would call "promiscuous" functions. Thus, promiscuity may engage highly conserved structural regions of the protein with dual roles, contributing to both a naturally evolved (native) and a promiscuous function. For instance, the flexibility of the beta-hairpin flap in HIV-1 protease is required for the processivity of the enzyme (6). Thus, the flap region, must have a highly water-exposed - and hence labile - hydrogen bond, as needed to confer the necessary flexibility associated with the gating mechanism. A naturally evolved and highly conserved glycine-rich loopy region exposes to water a backbone hydrogen bond in the P-hairpin. The over-exposed hydrogen bond is inherently sticky because it can be strengthened and stabilized upon exogenous water removal (6 - 8). Thus, the lack of protection on the flap backbone hydrogen bond is subservient to a native function of the HIV-1 protease, but becomes also the reason for its stickiness, a property taken advantage of in a promiscuous function. A proper inhibition of the protease then hinges upon the possibility of wrapping of the flap hydrogen bonds with the nonpolar groups of the purported drugs (6). In this way, we are reporting on an instance of a structural feature - a flexible flap - naturally selected for a purpose and utilized promiscuously for another purpose. Undeniably, the protein has actually evolved to sustain this feature, thus hinting to an apparent inconsistency in the views of Aharoni et al. (1), who maintain that promiscuous functions are not naturally evolved. Furthermore, there are naturally evolved and conserved structural features inherent to catalytic activity that have been used promiscuously by the drug designers aiming at the inhibition of HIV-1 protease activity. Thus, there are intramolecularly under-wrapped or under-dehydrated hydrogen bonds adjacent to the catalytically active site (Asp25) in each monomer of the functionally compentent homodimer (6). These structural features are required to frame an anchoring track for the substrate peptide. This "sticky track" determined by the under-dehydrated hydrogen bonds is required to align the substrate peptide chain across the cavity, as needed for selective nucleophilic attack by the two equivalent catalytic Asp25s. Furthermore, since such bonds promote the removal of surrounding water (6 - 8), they enhance the electrostatic field generated by the catalytic Asp25, by de-screening its net charge. This is precisely their raison d'etre: they foster catalytic activity by exacerbating the nucleophilic potential of Asp25. On the other hand, since these hydrogen bonds are inherently sticky for the reasons mentioned above, they have been targeted by drug designers aiming at inhibiting the protease activity (6). Thus, drug inhibitors provide intermolecular wrapping to these naturally evolved packing defects in the protease Here we find another instance of naturally evolved features compliant with a native catalytic function and used promiscuously for drug-based inhibition. These facts and the very nature of drug discovery seem to disprove the basic tenet that drug binding is quintessentially promiscuous because proteins did not evolve to associate with man-made ligands. In fact, every native function may be turned promiscuous by a sufficiently skillful designer of ligands able to mimic natural substrates and knowledgeable of the mechanisms of protein associations. On the other hand, few native functions may escape promiscuity because exogenous water removal from pre-formed electrostatics - an entropy-related interaction - is a ubiquitous determinant of protein associations (7, 8).
