Immucillins in custom catalytic-site cavities

Purine nucleoside phosphorylase (PNP)1 catalyzes the phosphorolytic cleavage of inosine and guanosine as well as their 2′-deoxy analogues to (deoxy)ribose 1-phosphate and hypoxanthine or guanine (Figure 1). Figure 1 Phosphorolysis of inosine catalyzed by PNP. Inhibition of human PNP activity causes accumulation of deoxyguanosine which in turn leads to downstream inhibition of cell division and apoptosis specifically in T-lymphocytes.2–4 Thus, PNP has been identified as a target for the treatment of T-cell lymphoma, rheumatoid arthritis, psoriasis, multiple sclerosis, and other T-cell mediated disorders.5 The human PNP-catalyzed reaction has been shown to proceed through a dissociative transition state characterized by a ribooxacarbenium ion with a cationic C-1′ which is separated from the nucleobase by > 3 A.6 These features were incorporated into a family of transition-state analogue inhibitors called Immucillins (Figure 2), whose members typically possess low nanomolar to picomolar affinity for human PNP. The potent inhibitors exhibit slow-onset behavior, where a time-dependent conformational change converts the initial enzyme-inhibitor complex (E·I), with dissociation constant Ki, to an even more stable complex (E*·I), with dissociation constant Ki*. The first-generation inhibitor, Immucillin-H (ImmH, 1),7 has a Ki of 3.3 nM and a Ki* of 58 pM.8 Modification of ImmH produced second-and third-generation Immucillins represented by DADMe-ImmH (4)9 and SerMe-ImmH (9),10 respectively, with Ki* values of 11 and 5.2 pM, respectively. Figure 2 Three generations of Immucillins, potent PNP transition-state analogue inhibitors. ImmH, DADMe-ImmH, and SerMe-ImmH are shown along with 5′-alkylthio and arylthio derivatives used in this study. Mutation of His257 has shown that this residue serves an important role in transition-state formation by hydrogen-bonding with the 5′-OH, orienting it into an electron-rich “oxygen stack” with O-4′ and OP from the phosphate nucleophile (Figure 3).8 This interaction is reasoned to provide electron density to weaken the ribosidic bond and stabilize the developing cationic transition state. Though mutation adversely affected the steady-state properties of PNP, mutants were capable of binding ImmH and DADMe-ImmH with reasonably good affinity. Structural analysis revealed that one of the mutants, His257Gly, was capable of binding these inhibitors nearly identically to the native protein, despite side-chain removal (Figure 4). We envisioned that the active-site cavity introduced in the His257Gly mutant could be exploited in the binding of bulkier Immucillin derivatives. Figure 3 Proposed role of His257 in formation of the transition state, featuring dynamic compression of the O5′–O4′–OP “oxygen stack”. The “oxygen stack” is represented by hashed bonds connecting ... Figure 4 Overlay of the crystal structures of native human PNP and His257Gly complexed with ImmH and phosphate (PO4). Side chains of selected active-site residues within 3.2 A of ImmH have been included. Carbon atoms of these residues and of ImmH are green ... 5′-Methylthio-ImmH (MeS-ImmH, 2) and 5′-phenylthio-ImmH (PhS-ImmH, 3)11 were originally developed as specific inhibitors of PNP from Plasmodium falciparum, exhibiting 112- and 2-fold binding preference over human PNP, respectively.12 With native human PNP, 2 and 3 bind with weaker affinity than 1, showing no slow-onset behavior and yielding Ki values of 101 and 160 nM, respectively (Table 1). In comparison to the substrate inosine (Km = 40 µM), modification of the 5′-hydroxyl of ImmH resulted in a drop in relative affinity (Km/Ki) from 690,000 to 400 and 250, respectively. Mutation of His257 to glycine abolished the slow-onset character of ImmH (1), resulting in a Ki of 11.0 nM. Taken in light of the elevated Km (750 µM) with this mutant, the relative affinity decreased tenfold relative to the native enzyme [(mutant Km/Ki)/(native Km/Ki) = 0.099]. In the case of the bulkier derivatives 2 and 3, however, not only was increased absolute affinity observed (Ki = 4.9 and 6.0 nM, respectively), the relative affinity increased to 150,000 and 120,000, respectively, up to 500-fold over native PNP. Table 1 Dissociation constants of transition-state analogues with native human PNP and His257Gly.a The second-generation transition-state analogue DADMe-ImmH (4) was designed to mimic the human PNP transition state by increasing the leaving group distance through introduction of a methylene bridge between the pseudoribosidic bond and by moving the cationic ring nitrogen to the 1′-position, where significant positive-charge character is developed at the transition state.13 With the native enzyme, DADMe- ImmH was found to be a more potent inhibitor than ImmH, giving a Ki* of 10.7 pM.8 Unlike the case with ImmH, the His257Gly mutant bound DADMe-ImmH in a slow-onset manner, with only slightly lowered relative affinity [(mutant Km/Ki)/(native Km/Ki) = 0.74]. 5′- Methylthio-DADMe-ImmH (MeS-DADMe-ImmH, 5) and 5′-propylthio-DADMe-ImmH (PrS-DADMe-ImmH, 6)12 maintained strong potency with the native enzyme with Ki* values of 19.6 pM (Km/Ki = 2,000,000) and 9.8 pM (Km/Ki = 4,100,000), respectively. These inhibitors were found to be strikingly effective with the His257Gly mutant, exhibiting slow-onset inhibition and yielding Ki* values of only 2.8 and 1.9 pM, respectively. Not only are these values lower than the most potent inhibitor tested to-date with native human PNP, the relative affinities (Km/Ki) of 270,000,000 and 400,000,000 are the largest ever reported for any enzyme-inhibitor system. Inhibitors 5 and 6 are therefore bound with 130- and 97-fold preference, respectively, over the native protein. Further derivatization of 5 to (±)-5′-deoxy-4′-fluoro-5′-methylthio-DADMe-ImmH (4′-F-MeS-DADMe-ImmH, 7) was accomplished by functional group exchange from the fluorinated diol precursor 11, followed by Mannich reaction of 13 with 9-deazahypoxanthine and formaldehyde (Scheme 1).14 Both the fluorinated derivative 7 and 5′-deoxy-4′-hydroxy-5′-methylthio-DADMe-ImmH (4′-OH-MeS-DADMe-ImmH, 8)12 resulted in the loss of slow-onset inhibition with native and mutant enzymes. These compounds bound nearly equally well to native PNP with relative affinities of 7,500 and 5,100, respectively. As in the case with 5 and 6, when compounds 7 and 8 were tested with the glycine mutant, the dissociation constants dropped, resulting in enhanced relative affinities of 540,000 and 600,000, respectively, corresponding to 78- and 120-fold improvements over the native enzyme. Scheme 1 Reagents and conditions: (a) Bu2SnO, toluene, reflux, then MsCl, 81%; (b) NaSMe, DMF, 68%; (c) 6N HCl; (d) 9-deazahypoxanthine, CH2O, NaOAc, water – dioxane, 100°C, 30%. The third generation of PNP transition-state inhibitors consist of acyclic analogues of DADMe-ImmH. SerMe-ImmH (9), despite its lack of stereocenters, binds to human PNP with a Ki* of 5.2 pM.10 As was observed with ImmH, mutation of His257 resulted in the loss of slow-onset behavior, lowering the relative affinity from 7,700,000 to 260,000. The corresponding ratio of the relative affinities is 0.033, which is the lowest among the ten inhibitors tested. SerMe-ImmH was also derivatized to the methylthio analogue 10 in a manner similar to that outlined for compound 7 above (Scheme 2).15 As observed for compounds 7 and 8, incubation of MeS-SerMe-ImmH (10) with native and mutant PNP lacked the slow-onset property of its underivatized analogue, yielding a lower Km/Ki with the native protein (9,300) but a larger value (680,000) with His257Gly. The discrimination for 10 by His257Gly is therefore 73-fold greater than that by native PNP. Scheme 2 Reagents and conditions: (a) Boc2O, MeOH; (b) 1 eq. NaH, TBDMSCl, THF, 71% (2 steps); (c) MsCl, Et3N, CH2Cl2; (d) NaSMe, DMF, 78% (2 steps); (e) HCl, MeOH, H2O; (f) 9-deazahypoxanthine, CH2O, NaOAc, H2O, 80°C, 28% (2 steps). This study has confirmed that human PNP tolerates substitution of the 5′-hydroxyl of the transition-state analogues ImmH, DADMe-ImmH, and SerMe-ImmH with alkylthio and arylthio groups; however, except for compounds 5 and 6, the slow-onset nature of inhibition is lost, and inhibitor dissociation constants increase by two to three orders of magnitude. When the imidazole moiety of residue 257 is removed by mutation, the loss of an H-bond partner for the 5′-OH likely accounts for the observed decreases in binding affinity for the unmodified analogues 1, 4, and 9. Nevertheless, the active-site cavity that is created accommodates bulkier functionalities which lack H-bonding opportunities. In all cases, the His257 mutant exhibited enhanced binding affinities relative to substrate for bulkier analogues over native PNP by factors ranging from 73 to 500. Unprecedented selective binding was observed with 5 and 6, which associate up to 400 million times more tightly with His257Gly than inosine.