Gary B. Evans

Third-Generation Immucillins: Syntheses and Bioactivities of Acyclic Immucillin Inhibitors of Human Purine Nucleoside Phosphorylase

ImmH (1) and DADMe-ImmH (2) are potent inhibitors of human purine nucleoside phoshorylase (PNP), developed by us and currently in clinical trials for the treatment of a variety of T-cell related diseases. Compounds 1 and 2 were used as templates for the design and synthesis of a series of acyclic immucillin analogues (8-38) in order to identify simplified alternatives to 1 and 2. SerMe-ImmG (8) and DATMe-ImmG (9) displayed the lowest inhibition constants of 2.1 and 3.4 pM, respectively, vs PNP. It was postulated that the flexible natures of 8 and 9 enabled them to adopt conformations resembling those of 1 and 2 within the active site of PNP and that the positioning of two hydroxyl groups was critical for picomolar activity. SerMe-ImmH (10, K(d) = 5.2 pM) was shown to be orally available in mice with a long biological residence time on blood PNP.

Azetidine based transition state analogue inhibitors of N-ribosyl hydrolases and phosphorylases.

N-ribosyl phosphorylases and hydrolases catalyze nucleophilic displacement reactions by migration of the cationic ribooxacarbenium carbon from the fixed purine to phosphate and water nucleophiles, respectively. As the lysis reaction progresses along the reaction coordinate, the distance between the purine and carbocation increases and the distance between carbocation and nucleophile decreases. Immucillin-H and DADMe-immucillin-H have been shown previously to be potent inhibitors of purine nucleoside phosphorylases and lie more toward the reactant and products side of this reaction coordinate, respectively. Both these enzyme inhibitors, which are currently in human clinical trials for different indications, are chiral and expensive to manufacture. We now report the synthesis of azetidine analogues of the DADMe-immucillins, which, despite their lack of stereochemical complexity, remain potent inhibitors (equilibrium dissociation constants as low as 229 pM) of purine nucleoside phosphorylase (PNP), methylthioadenosine phosphorylase (MTAP), and methylthioadenosine nucleosidase (MTAN), with potential utility as drug candidates.

Four generations of transition-state analogues for human purine nucleoside phosphorylase

Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (, first-generation) contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (, second-generation), uses a methylene-bridged dihydroxypyrrolidine cation with two asymmetric centers. DATMe-Immucillin-H (, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; 1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, 2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, 3) interaction between phosphate and inhibitor hydroxyl groups, and 4) His257 interacting with the 5′-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

Growth and Metastases of Human Lung Cancer Are Inhibited in Mouse Xenografts by a Transition State Analogue of 5′-Methylthioadenosine Phosphorylase

The S-adenosylmethionine (AdoMet) salvage enzyme 5′-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5′-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2−/−γC−/− and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP−/− and H358 MTAP+/+ tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.

Energetic Mapping of Transition State Analogue Interactions with Human and Plasmodium falciparum Purine Nucleoside Phosphorylases

Human purine nucleoside phosphorylase (huPNP) is essential for human T-cell division by removing deoxyguanosine and preventing dGTP imbalance. Plasmodium falciparum expresses a distinct PNP (PfPNP) with a unique substrate specificity that includes 5′-methylthioinosine. The PfPNP functions both in purine salvage and in recycling purine groups from the polyamine synthetic pathway. Immucillin-H is an inhibitor of both huPNP and PfPNPs. It kills activated human T-cells and induces purine-less death in P. falciparum. Immucillin-H is a transition state analogue designed to mimic the early transition state of bovine PNP. The DADMe-Immucillins are second generation transition state analogues designed to match the fully dissociated transition states of huPNP and PfPNP. Immucillins, DADMe-Immucillins and related analogues are compared for their energetic interactions with human and P. falciparum PNPs. Immucillin-H and DADMe-Immucillin-H are 860 and 500 pm inhibitors against P. falciparum PNP but bind human PNP 15–35 times more tightly. This common pattern is a result of kcat for huPNP being 18-fold greater than kcat for PfPNP. This energetic binding difference between huPNP and PfPNP supports the kchem/kcat binding argument for transition state analogues. Preferential PfPNP inhibition is gained in the Immucillins by 5′-methylthio substitution which exploits the unique substrate specificity of PfPNP. Human PNP achieves part of its catalytic potential from 5′-OH neighboring group participation. When PfPNP acts on 5′-methylthioinosine, this interaction is not possible. Compensation for the 5′-OH effect in the P. falciparum enzyme is provided by improved leaving group interactions with Asp206 as a general acid compared with Asn at this position in huPNP. Specific atomic modifications in the transition state analogues cause disproportionate binding differences between huPNP and PfPNPs and pinpoint energetic binding differences despite similar transition states.

Plasmodium falciparum Parasites Are Killed by a Transition State Analogue of Purine Nucleoside Phosphorylase in a Primate Animal Model

Plasmodium falciparum causes most of the one million annual deaths from malaria. Drug resistance is widespread and novel agents against new targets are needed to support combination-therapy approaches promoted by the World Health Organization. Plasmodium species are purine auxotrophs. Blocking purine nucleoside phosphorylase (PNP) kills cultured parasites by purine starvation. DADMe-Immucillin-G (BCX4945) is a transition state analogue of human and Plasmodium PNPs, binding with picomolar affinity. Here, we test BCX4945 in Aotus primates, an animal model for Plasmodium falciparum infections. Oral administration of BCX4945 for seven days results in parasite clearance and recrudescence in otherwise lethal infections of P. falciparum in Aotus monkeys. The molecular action of BCX4945 is demonstrated in crystal structures of human and P. falciparum PNPs. Metabolite analysis demonstrates that PNP blockade inhibits purine salvage and polyamine synthesis in the parasites. The efficacy, oral availability, chemical stability, unique mechanism of action and low toxicity of BCX4945 demonstrate potential for combination therapies with this novel antimalarial agent.

Inhibitors of ADP-Ribosylating Bacterial Toxins Based on Oxacarbenium Ion Character at Their Transition States

The bacterial exotoxins, cholera toxin (CT), pertussis toxin (PT), and diphtheria toxin (DT), interfere with specific host proteins to cause tissue damage for their respective infections. The common toxic mechanism for these agents is mono-ADP-ribosylation of specific amino acids in G(s)(alpha), G(i)(alpha), and eEF-2 proteins, respectively, by the catalytic A chains of the toxins (CTA, PTA, and DTA). In the absence of acceptor proteins, these toxins also act as NAD(+)-N-ribosyl hydrolases. The transition-state structures for NAD(+) hydrolysis and ADP-ribosylation reactions have oxacarbenium ion character in the ribose. We designed and synthesized analogues of NAD(+) to resemble their oxacarbenium ion transition states. Inhibitors with oxacarbenium mimics replacing the NMN-ribosyl group of NAD(+) show 200-620-fold increased affinity in the hydrolytic and N-ribosyl transferase reactions catalyzed by CTA. These analogues are also inhibitors for the hydrolysis of NAD(+) by PTA with K(i) values of 24-40 microM, but bind with similar affinity to the NAD(+) substrates. Inhibition of the NAD(+) hydrolysis and ADP-ribosyl transferase reactions of DTA gave K(i) values from 19 to 48 microM. Catalytic rate enhancements by the bacterial exotoxins are small, and thus transition-state analogues cannot capture large energies of activation. In the cases of DTA and PTA, analogues known to resemble the transition states bind with approximately the same affinity as substrates. Transition-state analogue interrogation of the bacterial toxins indicates that CTA gains catalytic efficiency from modest transition-state stabilization, but DTA and PTA catalyze ADP-ribosyl transferase reactions more from ground-state destabilization. pH dependence of inhibitor action indicated that both neutral and cationic forms of transition-state analogues bind to DTA with similar affinity. The origin of this similarity is proposed to reside in the cationic nature of NAD(+) both as substrate and at the transition state.

Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems

Summary Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

A picomolar transition state analogue inhibitor of MTAN as a specific antibiotic for Helicobacter pylori.

Campylobacter and Helicobacter species express a 6-amino-6-deoxyfutalosine N-ribosylhydrolase (HpMTAN) proposed to function in menaquinone synthesis. BuT-DADMe-ImmA is a 36 pM transition state analogue of HpMTAN, and the crystal structure of the enzyme-inhibitor complex reveals the mechanism of inhibition. BuT-DADMe-ImmA has a MIC(90) value of <8 ng/mL for Helicobacter pylori growth but does not cause growth arrest in other common clinical pathogens, thus demonstrating potential as an H. pylori-specific antibiotic.

Conformational States of Human Purine Nucleoside Phosphorylase at Rest, at Work, and with Transition State Analogues

Human purine nucleoside phosphorylase (PNP) is a homotrimer binding tightly to the transition state analogues Immucillin-H (ImmH; K(d) = 56 pM) and DATMe-ImmH-Immucillin-H (DATMe-ImmH; K(d) = 8.6 pM). ImmH binds with a larger entropic penalty than DATMe-ImmH, a chemically more flexible inhibitor. The testable hypothesis is that PNP conformational states are more relaxed (dynamic) with DATMe-ImmH, despite tighter binding than with ImmH. PNP conformations are probed by peptide amide deuterium exchange (HDX) using liquid chromatography high-resolution Fourier transform ion cyclotron resonance mass spectrometry and by sedimentation rates. Catalytically equilibrating Michaelis complexes (PNP.PO(4).inosine <--> PNP.Hx.R-1-P) and inhibited complexes (PNP.PO(4).DATMe-ImmH and PNP.PO(4).ImmH) show protection from HDX at 9, 13, and 15 sites per subunit relative to resting PNP (PNP.PO(4)) in extended incubations. The PNP.PO(4).ImmH complex is more compact (by sedimentation rate) than the other complexes. HDX kinetic analysis of ligand-protected sites corresponds to peptides near the catalytic sites. HDX and sedimentation results establish that PNP protein conformation (dynamic motion) correlates more closely with entropy of binding than with affinity. Catalytically active turnover with saturated substrate sites causes less change in HDX and sedimentation rates than binding of transition state analogues. DATMe-ImmH more closely mimics the transition of human PNP than does ImmH and achieves strong binding interactions at the catalytic site while causing relatively modest alterations of the protein dynamic motion. Transition state analogues causing the most rigid, closed protein conformation are therefore not necessarily the most tightly bound. Close mimics of the transition state are hypothesized to retain enzymatic dynamic motions related to transition state formation.