Molecular recognition of protein-ligand complexes : applications to drug design

FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP

https://www.pnas.org/content/pnas/95/18/10437.full.pdf

Redesigning an FKBP–ligand interface to generate chemical dimerizers with novel specificity

FK506 is a new FDA-approved immunosuppressant used for prevention of allograft rejection in, for example, liver and kidney transplantations. FK506 is inactive by itself and requires binding to an FK506 binding protein-12 (FKBP-12), or immunophilin, for activation. In this regard, FK506 is analogous to cyclosporin A, which must bind to its immunophilin (cyclophilin A) to display activity. This FK506-FKBP complex inhibits the activity of the serine/threonine protein phosphatase 2B (calcineurin), the basis for the immunosuppressant action of FK506. The discovery that immunophilins are also present in the nervous system introduces a new level of complexity in the regulation of neuronal function. Two important calcineurin targets in brain are the growth-associated protein GAP-43 and nitric oxide (NO) synthase (NOS). This review focuses on studies showing that systemic administration of FK506 dose-dependently speeds nerve regeneration and functional recovery in rats following a sciatic-nerve crush injury. The effect appears to result from an increased rate of axonal regeneration. The nerve regenerative property of this class of agents is separate from their immunosuppressant action because FK506-related compounds that bind to FKBP-12 but do not inhibit calcineurin are also able to increase nerve regeneration. Thus, FK506's ability to increase nerve regeneration arises via a calcineurin-independent mechanism (i.e., one not involving an increase in GAP-43 phosphorylation). Possible mechanisms of action are discussed in relation to known actions of FKBPs: the interaction of FKBP-12 with two Ca2+ release-channels (the ryanodine and inositol 1,4,5-triphosphate receptors) which is disrupted by FK506, thereby increasing Ca2+ flux; the type 1 receptor for the transforming growth factor-beta (TGF-beta 1), which stimulates nerve growth factor (NGF) synthesis by glial cells, and is a natural ligand for FKBP-12; and the immunophilin FKBP-52/FKBP-59, which has also been identified as a heat-shock protein (HSP-56) and is a component of the nontransformed glucocorticoid receptor. Taken together, studies of FK506 indicate broad functional roles for the immunophilins in the nervous system. Both calcineurin-dependent (e.g., neuroprotection via reduced NO formation) and calcineurin-independent mechanisms (i.e., nerve regeneration) need to be invoked to explain the many different neuronal effects of FK506. This suggests that multiple immunophilins mediate FK506's neuronal effects. Novel, nonimmunosuppressant ligands for FKBPs may represent important new drugs for the treatment of a variety of neurological disorders.

Fk506 and the role of immunophilins in nerve regeneration

The new immunosuppressant drug FK506 (Tacrolimus) increases the rate of nerve regeneration in vivo (Gold et al. , 1994;Gold et al. , 1995). In the present study, we have examined the dose-dependence of FK506’s ability to enhance nerve regeneration. In the first set of experiments, rats received daily s.c. injections of FK506 (2 mg/kg, 5 mg/kg or 10 mg/kg) for 18 days after a sciatic nerve crush injury. Signs of functional recovery in the hind feet appeared earlier than in saline-treated control rats at all three FK506 dosage; recovery was maximally accelerated in the 5-mg/kg group. Light microscopy at 18 days after nerve crush revealed more regenerating myelinated fibers in FK506-treated rats than in controls; this was most apparent in the 5-mg/kg group. Morphometric analysis of axonal areas in the soleus nerve confirmed that axonal calibers were maximally increased in the 5-mg/kg group. In the second set of experiments, the rate of axonal regeneration was determined by radiolabeling the L5 dorsal root ganglion. Regeneration rate for sensory axons was maximally increased (by 34%) in the 5-mg/kg group. In contrast, cyclosporin A (10 or 50 mg/kg; dosages were selected on the basis of the 1 10 lower potency of cyclosporin A) did not significantly alter the rate of axonal regeneration. Cyclosporin A (50 mg/kg) also failed to increase functional recovery or axonal calibers in the soleus nerve. Because the two drugs share a common mechanism for producing immunosuppression ( i.e. , calcineurin inhibition), these results indicate that FK506’s nerve regenerative property involves a distinct, calcineurin-independent mechanism.

Comparative dose-dependence study of fk506 and cyclosporin a on the rate of axonal regeneration in the rat sciatic nerve

A Nonimmunosuppressant FKBP-12 Ligand Increases Nerve Regeneration

We have synthesized a series of non-macrocyclic ligands to FKBP12 that are comparable in binding potency and peptidyl prolyl isomerase (PPIase) inhibition to FK506 itself. We have also solved the structure of one of these ligands in complex with FKBP12, and have compared that structure to the FK506–FKBP12 complex. Consistent with the observed inhibitory equipotency of these compounds, we observe a strong similarity in the conformation of the two ligands in the region of the protein that mediates PPIase activity. Our compounds, however, are not immunosuppressive. In the FKBP12–FK506 complex, a significant portion of the FK506 ligand, its `effector domain', projects beyond the envelope of the binding protein in a manner that is suggestive of a potential interaction with a second protein, the calcium-dependent phosphatase, calcineurin, whose inhibition by the FKBP 12–FK506 complex interrupts the T-cell activation events leading to immunosuppression. In contrast, our compounds bind within the surface envelope of FKBP12, and induce significant changes in the structure of the FKBP12 protein which may also affect calcineurin binding indirectly.

Design, synthesis and structure of non-macrocyclic inhibitors of FKBP12, the major binding protein for the immunosuppressant FK506.

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Design, synthesis and structure of non-macrocyclic inhibitors of FKBP12, the major binding protein for the immunosuppressant FK506.